Cloning and characterization of slc26a6, slc26a1, and slc26a2 anion exchangers

ABSTRACT

Isolated nucleic acids encoding SLC26A6, SLC26A1, and SLC26A2 anion transporter polypeptides, recombinantly expressed SLC26A6, SLC26A1, and SLC26A2 anion transporter polypeptides, heterologous expression systems for recombinant expression of SLC26A6, SLC26A1, and SLC26A2 anion transporter polypeptides, assay methods employing the same, and methods for modulation of anion transport activity.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is based on and claims priority to U.S. ProvisionalApplication Ser. No. 60/360,275, filed Feb. 28, 2002, and entitledCLONING AND CHARACTERIZATION OF SLC26A6, SLC26A1, and SLC26A2 ANIONEXCHANGERS, herein incorporated by reference in its entirety.

GRANT STATEMENT

This work was supported by grants RO1 DK57708, PO1 DK038226, RO1DK56218, and T32 DK07569-12 from the U.S. National Institute of Health.Thus, the U.S. government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention generally relates to anion transporterpolypeptides and anion transport activity mediated by the same. Moreparticularly, the present invention provides isolated nucleic acidsencoding SLC26 anion transporter polypeptides, isolated and functionalSLC26 anion transporter polypeptides, a heterologous expression systemfor recombinant expression of SLC26 anion transporter polypeptides,methods for identifying modulators of an anion transporter, and usesthereof. Table of Abbreviations AE - anion exchanger ATCC - AmericanType Culture Collection BAC - bacterial artificial chromosome BLAST -basic alignment and search tool CF - cystic fibrosis CFTR - cysticfibrosis transmembrane conductance regulator cM - centimorgan CMV -cytomegalovirus cRNA - complementary RNA CpG - unmethylatedcytosine-guanine dinucleotides DIDS -4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid DTST - diastrophicdysplasia sulphate transporter; SLC26A2 EGFP - enhanced greenfluorescent protein EST - expressed sequence tag Fab - antigen-bindingantibody fragment FCS - Fluorescence Correlation Spectroscopy Fv -antigen-binding antibody fragment GAPDH - glyceraldehyde-3-phosphatedehydrogenase GFP - green fluorescent protein HEPES -4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid HTGS - highthroughput genomic sequences HUGO - Human Genome OrganizationI.M.A.G.E. - Integrated Molecular Analysis of Genomes and theirExpression database LA-PCR - long and accurate PCR LDB - locationdatabase MGD - Mouse Genome Database MHC - major histocompatibilitycomplex mSLC26A6 - mouse SLC26A6 NMDG - N-methyl-D-glucamine ORF - openreading frame OSM - osmolarity PAC - P-1 derived artificial chromosomepCMV-SLC26 - construct encoding SLC26 under the control of a CMVpromoter PCR - polymerase chain reaction PFU - plaque-forming unitpH_(i) - intracellular pH PKA - phosphokinase A PKC - phosphokinase CRACE - rapid amplification of cDNA ends RH - radiation hybrid RT-PCR -reverse transcription - polymerase chain reaction Sat-1 - sulphate aniontransporter-1 (SLC26A1) SDS - sodium dodecyl sulphate SELDI-TOF -Surface-Enhanced Laser Desorption/Ionization Time-Of-flight SpectroscopySLC26 - solute carrier 26 protein family Sp1 - pregnancy-“specific” beta1-glycoprotein; Cys₂-His₂ zinc finger transcription factor SPR - surfaceplasmon resonance STAS - sulfate transporter and anti-sigma domain STS -sequence-tagged site TESS - Transcription Element Search Software UTR -untranslated region V_(m) - membrane voltage xSLC26A1 - Xenopus SLC26A1ortholog xSLC26A6 - Xenopus SLC26A6 ortholog xPDS1 - Xenopus SLC26A4(pendrin or PDS) ortholog xPDS2 - Xenopus SLC26A4 (pendrin or PDS)ortholog xPDS3 - Xenopus SLC26A4 (pendrin or PDS) ortholog

BACKGROUND OF THE INVENTION

Anion exchange at the plasma membrane is primarily mediated by theproducts of two structurally distinct gene families: (1) the AE (anionexchanger) genes, which form a subset of the bicarbonate transporterSLC4 superfamily (Romero et al., 2000; Tsuganezawa et al., 2001); and(2) the SLC26 or sulphate permease gene family (Everett & Green, 1999).Members of the SLC26 gene family have been identified by expressioncloning (Bissig et al., 1994), subtractive cDNA cloning (Zheng et al.,2000), and positional cloning of human disease genes (Everett & Green,1999).

The SLC26 gene family has been highly conserved during evolution, andhomologues have been identified in bacteria, yeast, plants, and animals.See Everett & Green (1999) Hum Mol Genet 8:1883-1891 and Kere et al.(1999) Am J Physiol 276:G7-G13. Four mammalian SLC26 genes have beendescribed (SLC26A1, SLC26A2, SLC26A3, and SLC26A4). The Drosophilagenome contains at least nine family members, suggesting that additionalmammalian paralogues also exist.

Physiological roles for individual family members includetransepithelial salt transport (Everett & Green, 1999; Scott & Karniski,2000), thryoidal iodide transport (Scott et al., 1999), development andfunction of the inner ear (Everett & Green, 1999; Zheng et al., 2000),sulphation of extracellular matrix (Satoh et al., 1998), and renalexcretion of bicarbonate (Royaux et al., 2001) and oxalate (Karniski etal., 1998a). The various substrates transported by the SLC26 anionexchangers include sulphate (SO₄ ²⁻), chloride (Cl⁻), iodide (I⁻),formate, oxalate, hydroxyl ion (OH⁻), and bicarbonate (HCO₃ ⁻) (Bissiget al., 1994; Karniski et al., 1998a; Satoh et al., 1998; Moseley etal., 1999; Scott & Karniski, 2000; Soleimani et al., 2001).

The multiple physiological roles of SLC26 transporters are supported bydiverse anion transport properties. Despite a capacity for versatileanion exchange, SLC26 anion transporters display distinct patterns ofanion specificity and cis-inhibition. For example, SLC26A4, also knownas pendrin, can transport chloride, hydroxyl ion, bicarbonate, iodide,and formate, but neither oxalate nor sulphate (Scott et al., 1999; Scott& Karniski, 2000; Royaux et al., 2001; Soleimani et al., 2001).

Thus, there exists a long-felt need in the art to identify andfunctionally characterize SLC26 anion transporters as pharmaceuticaltargets for diseases and disorders related to abnormal anion transportactivity.

To meet this need, the present invention provides functional SLC26A6,SLC26A1, and SLC26A2 anion transporter polypeptides. The presentinvention also provides methods for identifying and using modulators ofanion transport via SLC26A6, SLC26A1, and SLC26A2.

SUMMARY OF INVENTION

The present invention provides isolated and functional SLC26A6polypeptides useful in the assays and screening methods disclosedherein. A functional SLC26A6 polypeptide can comprise: (a) a polypeptideencoded by a nucleic acid of any one of odd-numbered SEQ ID NOs:1-7; (b)a polypeptide encoded by a nucleic acid substantially identical to anyone of odd-numbered SEQ ID NOs:1-7; (c) a polypeptide comprising anamino acid sequence of any one of even-numbered SEQ ID NOs:2-8; or (d) apolypeptide substantially identical to any one of even-numbered SEQ IDNOs:2-8.

A functional SLC26A6 polypeptide can also comprise a polypeptide encodedby an isolated nucleic acid molecule selected from the group consistingof: (a) an isolated nucleic acid molecule encoding a polypeptide of anyone of even-numbered SEQ ID NOs:2-8; (b) an isolated nucleic acidmolecule of any one of odd-numbered SEQ ID NOs:1-7; (c) an isolatednucleic acid molecule which hybridizes to a nucleic acid of any one ofodd-numbered SEQ ID NOs:1-7 under wash stringency conditions representedby a wash solution having less than about 200 mM salt concentration anda wash temperature of greater than about 45° C., and which encodes afunctional SLC26A6 polypeptide; and (d) an isolated nucleic acidmolecule differing by at least one functionally equivalent codon fromthe isolated nucleic acid molecule of one of (a), (b), and (c) above innucleic acid sequence due to the degeneracy of the genetic code, andwhich encodes a functional SLC26A6 polypeptide encoded by the isolatednucleic acid of one of (a), (b), and (c) above.

Preferably, a functional property of a SLC26A6 polypeptide of theinvention comprises Cl⁻-fomate exchange, Cl⁻—Cl⁻ exchange, SO₄ ²⁻exchange, Cl⁻-oxalate exchange, Cl⁻-base exchange, or combinationsthereof. Cl⁻-base exchange is preferably electrogenic and can utilizesubstrates such as HCO₃ ⁻.

The present invention also provides isolated human SLC26A6apolypeptides, and nucleic acids encoding the same. Preferably, a humanSLC26A6a polypeptide comprises: (a) a polypeptide of SEQ ID NO:2; or (b)a polypeptide encoded by a nucleic acid of SEQ ID NO:1.

The present invention further provides isolated mouse SLC26A6polypeptides, SLC26A1 polypeptides, and nucleic acids encoding the same.Preferably, a mouse SLC26A6 polypeptide comprises: (a) a polypeptide ofSEQ ID NO:6 or 8; or (b) a polypeptide encoded by a nucleic acid of SEQID NO:5 or 7. Preferably, a mouse SLC26A1 polypeptide comprises: (a) apolypeptide of SEQ ID NO:10; or (b) a polypeptide encoded by a nucleicacid of SEQ ID NO:9.

Also provided are systems for recombinant expression of a SLC26polypeptide. The system comprises: (a) a SLC26 polypeptide of theinvention (representative embodiments set forth as SEQ ID NOs:2, 6, 8,and 10); and (b) a host cell expressing the SLC26 polypeptide. A hostcell can comprise any suitable cell. A preferred host cell comprises amammalian cell, more preferably a human cell.

Using the disclosed system for recombinant expression of a SLC26polypeptide, the present invention further provides a method foridentifying modulators of anion transport. Also provided are modulatorsof anion transport that are identified by the disclosed methods.

In a preferred embodiment of the invention a method for identifying amodulator of anion transport comprises: (a) providing a recombinantexpression system whereby a functional SLC26 polypeptide is expressed ina host cell, and wherein the SLC26 polypeptide comprises a humanSLC26A6a polypeptide, a mouse SLC26A6 polypeptide, or a mouse SLC26A1polypeptide; (b) providing a test substance to the system of (a); (c)assaying a level or quality of SLC26 function in the presence of thetest substance; (d) comparing the level or quality of SLC26 function inthe presence of the test substance with a control level or quality ofSLC26 function; and (e) identifying a test substance as an aniontransport modulator by determining a level or quality of SLC26 functionin the presence of the test substance as significantly changed whencompared to a control level or quality of SLC26 function.

In another embodiment of the invention, a method for identifying amodulator of anion transport comprises: (a) exposing a SLC26Apolypeptide to one or more test substances, wherein the SLC26Apolypeptide comprises a human SLC26A6a polypeptide, a mouse SLC26A6polypeptide, or a mouse SLC26A1 polypeptide; (b) assaying binding of atest substance to the isolated SLC26A6 polypeptide; and (c) selecting acandidate substance that demonstrates specific binding to the SLC26A6polypeptide.

The present invention further provides methods for modulating aniontransport activity in a subject. Preferably, the subject is a mammaliansubject, and more preferably a human subject. Also preferably, the aniontransport activity that is altered in a subject comprises an activity ofa SLC26A6 polypeptide.

In one embodiment of the present invention, a method for modulatinganion transport activity in a subject comprises: (a) preparing acomposition comprising a SLC26 modulator identified according to thedisclosed methods, and a pharmaceutically acceptable carrier; (b)administering an effective dose of the composition to a subject, wherebyanion transport activity in the subject is altered.

The present invention further provides a method for activating a SLC26A1polypeptide in a subject via administering a SLC26A1 modulator to thesubject, wherein the SLC26A1 modulator comprises an impermeant anionsuch as Cl⁻ or formate.

Also provided is a method for modulating a SLC26A2 polypeptide or aSLC26A6 polypeptide in a subject by administering a pH modifier.

Accordingly, it is an object of the present invention to provide novelSLC26 nucleic acids and polypeptides, heterologous expression systemswhereby a SLC26 polypeptide is expressed, methods and assays employing aheterologous SLC26 expression system, and methods for modulating anddetecting a SLC26 polypeptide. This object is achieved in whole or inpart by the present invention.

An object of the invention having been stated above, other objects andadvantages of the present invention will become apparent to thoseskilled in the art after a study of the following description of theinvention, Figures, and non-limiting Examples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an alignment of a conserved SLC26 domain encompassing theProsite “sulfate transport” signature sequence (Bucher & Bairoch, 1994;Hofmann et al., 1999) (http://www.expasy.ch/prosite/) in mouse SLC26A1(SEQ ID NO:70), mouse SLC26A2 (SEQ ID NO:71), mouse SLC26A3 (SEQ IDNO:72), mouse SLC26A4 (SEQ ID NO:73), mouse SLC26A5 (SEQ ID NO:74),mouse SLC26A6 (SEQ ID NO:75), mouse SLC26A7 (SEQ ID NO:76), mouseSLC26A8 (SEQ ID NO:77), mouse SLC26A9 (SEQ ID NO:78), and mouse SLC26A11(SEQ ID NO:79). The 22-residue Prosite motif is underlined in sequencesthat conform to the consensus (SLC26A1, SLC26A2, SLC26A3, and SLC26A11).Shading, similar residues, conservative substitutions, and weaklysimilar residues; asterisks (*), invariant residues.

FIG. 2 is an alignment of the first 100 amino acids of mouse and humanSLC26A6a proteins (SEQ ID NOs:6 and 2, respectively). Boxed sequences,unique amino-terminal extensions predicted in the longer SLC26A6aproteins (when compared to SLC26A6b proteins); asterisk (*), predictedPKC phosphorylation sites.

FIG. 3 is the predicted sequence of mouse SLC26A1 protein (SEQ IDNO:10). (●) N-glycosylation sites; asterisk (*), PKC sites; (♦) PKAsites; (▪) combined PKC/PKA sites; (▴) tyrosine kinase sites; underline,potential transmembrane domains.

FIG. 4 presents the sequence of the proximal promoter of the mouseSLC26A6 gene (SEQ ID NO:13). Coding sequence from the 3′ end of exon 1ais underlined. A predicted CpG island includes the sequence betweenbrackets ([ ]). Potential binding sites for transcription factors areboxed and labeled. The binding sites were predicted using the TESS(Schug & Overton, 1997); available at http://www.cbil.upenn.edu/tess)and Matinspector (Quandt et al., 1995); available from GenomatixSoftware GmbH of Munich Germany) programs. C/EBPbeta,CCAAT/enhancer-binding protein beta isoform; E12/E47, E2A immunoglobulinenhancer binding factors; Sp1, Sp1 transcription factors; AP-2, enhancerbinding protein AP-2; GATA-1, GATA-1 transcription factor; GR,glucocorticoid receptor; PR, progesterone receptor; AP-4, enhancerbinding factor AP-4; CP-2, chromosomal protein 2; NF-KB, NF kappa Btranscription factor; AP-1, enhancer binding protein AP-1; MAF, c-maftranscription factor.

FIGS. 5A-5D depict the expression of human and mouse SLC26A6.

FIG. 5A is a Northern blot prepared using the human tissues indicated.The blot was hybridized with a probe designed according to sequence atthe 3′ end of the SLC26A6 cDNA. Numbers at left indicate transcript sizein kD.

FIG. 5B is a Northern blot prepared using human pancreatic (Panc-1) andpulmonary (Calu-3) cell lines. The blot was hybridized with a probedesigned according to sequence at the 3′ end of the SLC26A6 cDNA.Numbers at left indicate transcript size in kD.

FIG. 5C is a Northern blot prepared using the mouse tissues indicated.The blot was hybridized with a probe designed according to sequence atthe 3′ end of the SLC26A6 cDNA. Numbers at left indicate transcript sizein kD.

FIG. 5D is a picture of a 6% acrylamide gel showing resolution ofSLC26A6 RT-PCR products. RT-PCR amplification of mouse SLC26A6 wasperformed using a sense primer in exon 1a and an anti-sense primer inexon 4. The reactions included a water template (H₂O), intestine RNA,heart RNA, and lung RNA. An additional control reaction was performed inwhich the reverse transcription step was omitted (RT(−)). The SLC26A6atranscript yields a 300 base pair product due to alternative splicing ofthe 5′ exon 1b. The SLC26A6b transcript yields a 438 base pair productby retention of exon 1b. Both transcripts are detected in intestine,heart, and lung, but not in water and no reverse transcription controls.Numbers at left indicate amplification product size in base pairs (bp).

FIGS. 6A and 6B depict anion transport activity of mouse SLC26A6b.

FIG. 6A is a bar graph that presents DIDS (1 mM)-sensitive ³⁵SO₄ ²⁻uptake (pmoVoocyte/h) in oocytes expressing SLC26A6 or SLC26A1. Controloocytes (H₂O) expressed neither SLC26A6 nor SLC26A1. Open bars,extracellular pH 7.4; Solid bars, extracellular pH 6.0; asterisk (*),statistically significant difference (p<0.01) when compared towater-injected oocytes; h, hour.

FIG. 6B is a bar graph that presents ³⁶Cl⁻ uptake (pmol/oocyte/h) inoocytes expressing SLC26A6 or SLC26A1. Control cells (H₂O) expressedneither SLC26A6 nor SLC26A 1. A second group of oocytes expressingSLC26A 1 were incubated in 25 mM SO₄ ²⁻ during the uptake in an attemptto stimulate Cl⁻ exchange (SLC26A1, SO₄). Open bars, extracellular pH7.4; Solid bars, extracellular pH 6.0; h, hour.

FIG. 6C is a bar graph depicting a differential effect of extracellularCl⁻ on ³⁵SO₄ ²⁻ uptake. Oocytes expressing SLC26A1, oocytes expressionSLC26A6, or control oocytes (H₂O) were incubated in medium containing³⁵SO₄ ²⁻ for one hour. Open bars, Cl⁻-free medium; solid gray bars, 25mM Cl⁻ added to the medium.

FIG. 6D is a bar graph depicting an effect of pH on ³⁵SO₄ ²⁻ uptake.Oocytes expressing SLC26A 1, oocytes expression SLC26A6, or controloocytes (H₂O) were incubated in medium containing ³⁵SO₄ ²⁻ and 25 mMCl⁻. Open bars, pH 7.4; solid bars, pH 6.0.

FIGS. 7A-7C are bar graphs that summarize cis-inhibition of Cl⁻ and SO₄²⁻ transport mediated by SLC26A6.

FIG. 7A is a bar graph that depicts cis-inhibition of Cl⁻—Cl⁻ exchangein oocytes expressing SLC26A6 or in control oocytes (H₂O). Oocytes wereincubated in medium containing ³⁶Cl⁻ for one hour in the absence(control) or presence of 25 mM of the indicated anions.

FIG. 7B is a bar graph that depicts cis-inhibition of SO₄ ²⁻ exchange.Oocytes were incubated in medium containing ³⁵SO₄ ²⁻ for one hour in theabsence (control) or presence of 25 mM of the indicated anions.

FIG. 7C is a bar graph that depicts trans-stimulation of SO₄ ²⁻exchange. Oocytes were incubated in medium containing ³⁵SO₄ ²⁻ for onehour, washed three times with cold uptake medium, and then incubated for30 minutes in the absence (control) or presence of 10 mM of theindicated anions to stimulate ³⁵SO₄ ²⁻ efflux.

FIGS. 8A and 8B depict oxalate and formate transport mediated by SLC26A1and SLC26A6.

FIG. 8A is a bar graph showing oxalate uptake (pmol/oocyte/h) in oocytesexpressing SLC26A1 or SLC26A6. Control oocytes (H₂O) expressed neitherSLC26A6 nor SLC26A1. Asterisk (*), statistically significant difference(p<0.01) when compared to water-injected oocytes; h, hour.

FIG. 8B is a bar graph showing oxalate uptake (pmol/oocyte/h) in oocytesexpressing SLC26A1 or SLC26A6. Control oocytes (H₂O) expressed neitherSLC26A6 nor SLC26A1. Asterisk (*), statistically significant difference(p<0.01) when compared to water-injected oocytes; h, hour.

FIGS. 9A and 9B present a differential effect of extracellular anions onSO₄ ²⁻ and oxalate uptake by SLC26A1 and SLC26A6.

FIG. 9A is a bar graph showing ³⁵SO₄ ²⁻ uptake in oocytes expressingSLC26A 1 or in control oocytes (H₂O). Oocytes were incubated in mediumcontaining ³⁵SO₄ ²⁻ for one hour in the absence (control) or presence of25 mM of the indicated anions. Monovalent anions were observed toactivate ³⁵SO₄ ²⁻ transport by SLC26A1, whereas they inhibit ³⁵SO₄ ²⁻transport by SLC26A6 (FIG. 7B).

FIG. 9B is a bar graph showing oxalate uptake in oocytes expressingSLC26A1, oocytes expressing SLC25A6, or in control oocytes (H₂O).Oocytes were incubated in medium containing oxalate for one hour in theabsence (control) or presence of 25 mM of the indicated anions.Monovalent anions activated oxalate transport by SLC26A1, whereas theyinhibited oxalate transport by SLC26A6. Open bars, oocytes expressingSLC26A 1; solid bars, oocytes expressing SLC26A6.

FIGS. 10A-10C summarize anion transport activity of SLC26A2.

FIG. 10A is a bar graph showing ³⁵SO₄ ²⁻ uptake in oocytes expressingSLC26A2 or in control oocytes (H₂O). Oocytes were incubated in mediumcontaining ³⁵SO₄ ²⁻ for one hour in the absence of extracellular Cl⁻.Open bars, extracellular pH 7.4; Solid bars, extracellular pH 6.0; h,hour.

FIG. 10B is a bar graph showing that sulphate uptake by SLC26A2-injectedoocytes is cis-inhibited by extracellular Cl⁻. ³⁵SO₄ ²⁻ uptake wasmeasured for one hour in oocytes expressing SLC26A2 or in controloocytes (H₂O). (−) chloride-free media; (+) media containing 25 mM Cl⁻.

FIG. 10C is a bar graph that presents ³⁶Cl⁻ uptake (pmol/oocyte/h) inoocytes expressing SLC26A2 or control cells (H₂O). Open bars,extracellular pH 7.4; Solid bars, extracellular pH 6.0; h, hour.

FIGS. 11A-11B present a functional characterization of SLC26A6 usingion-selective microelectrodes.

FIGS. 11A-11B and 12A-12B present the results of electrophysiologicalexperiments described in Example 7.

FIG. 13 depicts chloride uptake in Xenopus oocytes expressing XenopusxPDS2, human SLC26A3 (DRA), mouse Slc26a6, and human SLC26A6, withextracellular pH of 7.5 (open bars) or 6.0 (dark fill). Chloride uptakeis significantly higher than that of water-injected oocytes (H₂O).

FIG. 14 depicts sulphate transport mediated by Xenopus oocytesexpressing mouse Slc26a6, at varying concentrations of extracellulars042- and constant amounts of tracer ³⁵SO₄ ²⁻.

FIG. 15 depicts sulphate transport mediated by Xenopus oocytesexpressing human SLC26A6, at varying concentrations of extracellular SO₄²⁻ and constant amounts of tracer ³⁵SO₄ ²⁻. Note the scale of theY-axis; absolute transport rates are much lower than in oocytesexpressing mouse Slc26a6 (FIG. 14).

FIG. 16 is a phylogenetic tree encompassing the ten murine Slc26proteins and the five Xenopus laevis xSLC26 proteins. The three xPOSproteins are most homologous to Slc26a4 (Pendrin or PDS), whereasxSLC26A1 and xSLC26A6 are the clear orthologs of murine Slc26a1 andSlc26a6, respectively.

FIG. 17 depicts chloride transport mediated by oocytes expressing fourof the Xenopus laevis xSLC26 anion exchangers; uptakes are significantlyhigher than that of water-injected oocytes (H₂O).

FIG. 18 depicts HCO₃ ⁻ transport mediated by xPDS2, characterized usingion-selective micro-electrodes. An experiment monitoring intracellularpH (pH_(i)) and membrane potential (V_(m)) of an xPDS2 oocyte is shown.The initial pH and rate of CO₂-induced acidification is equivalent tothat of the water control. In the continuing presence of 5% CO₂/33 mMHCO₃ ⁻ (pH 7.5), Cl⁻ removal elicits a robust alkalinization that haltswith Cl⁻ re-addition. Simultaneously, there is a pronounced andreversible depolarization not observed in control oocytes. Replacementof Na⁺ (choline) elicits no ΔpH_(i) and a small hyperpolarization asobserved in control cells.

FIG. 19 depicts cis-inhibition of ³⁶Cl⁻ uptake by various anions inoocytes expressing human SLC26A3 (DRA). Oocytes were exposed to 10 mMconcentrations of the anions noted during the uptake period; uptakemedium for the control group did not contain anions other than ³⁶Cl⁻ andgluconate.

FIG. 20 depicts Western blotting of oocyte lysates containing theindicated SLC26 proteins, using a 1:300 titre of a C-terminalSlc26a6-specific antibody; only the core (˜85 kDa) and glycosylated(˜100 kDa) SLC26A6 and Slc26a6 proteins are detected.

FIG. 21 depicts Western blotting of oocyte lysates containing theindicated SLC26 proteins, using a 1:300 titre of an N-terminalSlc26a6-specific antibody; only the core (˜85 kDa) and glycosylated(˜100 kDa) SLC26A6 and Slc26a6 proteins are detected.

BRIEF DESCRIPTION OF SEQUENCES IN THE SEQUENCE LISTING

Odd-numbered SEQ ID NOs:1-11 are nucleotide sequences described inTable 1. Even-numbered SEQ ID NOs:2-12 are protein sequences encoded bythe immediately preceding nucleotide sequence, e.g., SEQ ID NO:2 is theprotein encoded by the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:4is the protein encoded by the nucleotide sequence of SEQ ID NO:3, etc.

SEQ ID NO:13 is the mouse SLC26A6 promoter region.

SEQ ID NOs:14-55 are splice donor and acceptor sites of mouse SLC26A6,which are presented in Table 2.

SEQ ID NOs:56-61 are splice donor and acceptor sites of mouse SLC26A 1,which are presented in Table 3.

SEQ ID NOs:62-67 are primers.

SEQ ID NO:68 is a SLC26A6 conserved domain.

SEQ ID NO:69 is a SLC26 conserved domain.

SEQ ID NOs:70-79 are the SLC26 sequences indicated in Table 1, eachsequence encompassing the Prosite “sulphate transport” signaturesequence (Bucher & Bairoch, 1994; Hofmann et al., 1999)(http://www.expasy.ch/prosite/).

SEQ ID NOs:80-85 are the nucleic acid and amino acid sequences indicatedin Table 1 of three apparent orthologs of SLC26A4 (PDS1-3) isolated fromXenopus laevis.

SEQ ID NOs:86-87 are the nucleic acid and amino acid sequences,respectively, indicated in Table 1 of SLC26A1 isolated from Xenopuslaevis.

SEQ ID NOs:88-89 are the nucleic acid and amino acid sequences,respectively, indicated in Table 1 of SLC26A6 isolated from Xenopuslaevis.

SEQ ID NOs:90-91 are the nucleic acid and amino acid sequences,respectively, indicated in Table 1 of SLC26A6a isolated from pig (Susscrofa).

SEQ ID NO:92 is an amino acid sequence corresponding to residues 40-56of the mouse SLC26A6a protein.

SEQ ID NO:93 is an amino acid sequence corresponding to residues 631-649of the mouse SLC26A6a protein.

SEQ ID NO:94 is an amino acid sequence corresponding to residues 564-580of the mouse SLC26A1 protein.

SEQ ID NO:95 is an amino acid sequence corresponding to residues 6-22 ofthe human SLC26A2 protein.

SEQ ID NO:96 is an amino acid sequence for a human SLC26A2 polypeptide.TABLE 1 Sequence Listing Summary SEQ ID NO. Description 1-2 humanSLC26A6a 3-4 human SLC26A6b 5-6 mouse SLC26A6a 7-8 mouse SLC26A6b  9-10mouse SLC26A1 11-12 mouse SLC26A2 13 mouse SLC26A6 promoter region 14-55mouse SLC26A6 splice sites 56-61 mouse SLC26A1 splice sites 62-67primers 68 SLC26A6 conserved domain 69 SLC26 conserved domain 70 mouseSLC26A1 sulphate transport motif 71 mouse SLC26A2 sulphate transportmotif 72 mouse SLC26A3 sulphate transport motif 73 mouse SLC26A4sulphate transport motif 74 mouse SLC26A5 sulphate transport motif 75mouse SLC26A6 sulphate transport motif 76 mouse SLC26A7 sulphatetransport motif 77 mouse SLC26A8 sulphate transport motif 78 mouseSLC26A9 sulphate transport motif 79 mouse SLC26A11 sulphate transportmotif 80-81 Xenopus PDS1 82-83 Xenopus PDS2 84-85 Xenopus PDS3 86-87Xenopus SLC26A1 88-89 Xenopus SLC26A6 90-91 pig SLC26A6a 92 residues40-56 of the mouse SLC26A6a protein 93 residues 631-649 of the mouseSLC26A6a protein 94 residues 564-580 of the mouse SLC26A1 protein 95residues 6-22 of the human SLC26A2 protein 96 human SLC26A2

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

While the following terms are believed to be well understood by one ofordinary skill in the art, the following definitions are set forth tofacilitate explanation of the invention.

The terms “a,” and “an,” and “the” are used in accordance withlong-standing convention to refer to one or more.

The term “about”, as used herein when referring to a measurable valuesuch as a percentage of sequence identity (e.g., when comparingnucleotide and amino acid sequences as described herein below), anucleotide or protein length, an uptake amount, a pH value, etc. ismeant to encompass variations of ±20% or ±10%, more preferably ±5%, evenmore preferably ±1%, and still more preferably ±0.1% from the specifiedamount, as such variations are appropriate to perform a disclosed methodor otherwise carry out the present invention.

II. SLC26 Nucleic Acids and Polypeptides

The present invention provides novel SLC26 nucleic acids and novel SLC26polypeptides, including functional SLC26 polypeptides. The term “SLC26A”and terms including “SLC26” (e.g., SLC26A6, SLC26A1, and SLC26A2) refergenerally to isolated SLC26 nucleic acids, isolated polypeptides encodedby SLC26 nucleic acids, and activities thereof. SLC26 nucleic acids andpolypeptides can be derived from any organism.

The term “isolated”, as used in the context of a nucleic acid orpolypeptide, indicates that the nucleic acid or polypeptide exists apartfrom its native environment and is not a product of nature. An isolatednucleic acid or polypeptide can exist in a purified form or can exist ina non-native environment such as a transgenic host cell.

The terms “SLC26” and terms including “SLC26” also refer to polypeptidescomprising Na⁺-independent anion transporters that transport SO₄ ²⁻,Cl⁻, formate, and/or oxalate, and to nucleic acids encoding the same.

A region within the central hydrophobic core of SLC26 polypeptidesincludes a 22-residue “sulphate transport” consensus signature, Prositemotif PS01130 (Bucher & Bairoch, 1994; Hofmann et al., 1999)(http://www.expasy.ch/prosite/), which was initially defined bycomparison of the first mammalian family members with homologues inlower organisms. An alignment of this region is presented in FIG. 1.SLC26A6 functions as a sulphate transporter (Example 6), despite itslack of a consensus “sulphate transport” sequence, and thus thefunctional significance of this sequence motif is unclear. Within thisregion, many of the SLC26 proteins also share the sequence -GTSRHISV-(SEQ ID NO:69), whereas mouse SLC26A8 and mouse SLC26A9 depart from thisconsensus. The Prosite sulphate transport region also contains a totalof seven invariant residues, which are likely play a role in aniontransport (FIG. 1).

There is a second cluster of invariant residues at the C-terminal end ofthe hydrophobic core, in a conserved area defined by Saier et al (Saieret al., 1999). This region includes the triplet -NQE-, residues 417-419of mouse SLC26A6, which is conservatively variable only in SLC26A8(-NQD-). Three invariant residues in this section, E419, N425, and L483in mouse SLC26A2, have been shown to have functional significance inSHST1, a SLC26 homologue from the plant S. hamata (Khurana et al.,2000). Moreover, two of these invariant residues are mutated (N425D andL483P) in patients with a severe defect in human SLC26A2, causingachondrogenesis type 1B and/or atelosteogenesis type 2. The SLC26A2N425D mutant has further been shown to be non-functional in Xenopusoocytes (Karniski, 1989).

The C-terminal cytoplasmic domain of SLC26 proteins encompasses the STAS(Sulphate Transporter and Anti-Sigma) domain, recently defined by thehomology between the SLC26 proteins and bacterial anti-sigma factorantagonists (Aravind & Koonin, 2000). Structural features of this domainhave been predicted from the NMR analysis of the anti-sigma factorSPOIIAA (Aravind & Koonin, 2000), and include a characteristic α-helicalhandle. There is also a highly conserved loop interspersed between aβ-pleated sheet and α-helix, just upstream of the α-helical handle. Thisloop and β-pleated sheet have been proposed to play a role in nucleotidebinding and hydrolysis, in analogy to the known biochemistry of theanti-sigma factor antagonists (Aravind & Koonin, 2000). The loop ishighly conserved in SLC26 proteins and contains two invariant residues,D660 and L667 of mouse SLC26A2.

The STAS domain also contains a highly variable loop just proximal tothe β-pleated sheet and putative nucleotide binding loop (Aravind &Koonin, 2000). This variable loop is the site of significant insertionsin SLC26 proteins. The largest known insertion comprises 150 amino acidsin the case of human SLC26A8. Interestingly, no such insertion ispresent in bacterial SLC26 homologues, and this loop is the shortest inSLC26A11, which is arguably the most primeval of the mammalian SLC26paralogs.

The present invention provides novel human SLC26A6a polypeptides, whichis the shorter of two isoforms encoded by SLC26A6 and contains a uniqueamino-terminal extension. SLC26A6b is the longer isoform and lacks theamino-terminal extension. Also provided are novel nucleic acids encodinga human SLC26A6a polypeptide. A representative SLC26A6a nucleic acid ofthe present invention is set forth as SEQ ID NO:1, which encodes aSLC26A6a polypeptide set forth as SEQ ID NO:2.

The present invention further provides novel mouse SLC26A6 polypeptides,including SLC26A6a and SLC26A6b isoforms, and nucleic acids encoding thesame. Representative mouse SLC26A6a and mouse SLC26A6b nucleic acids areset forth as SEQ ID NOs:3 and 5, respectively. Representative mouseSLC26A6a and mouse SLC26A6b polypeptides are set forth as SEQ ID NOs:4and 6, respectively.

Also provided are novel mouse SLC26A1 polypeptides and nucleic acidsencoding the same. A representative SLC26A1 nucleic acid is set forth asSEQ ID NO:9, which encodes a SLC26A1 polypeptide set forth as SEQ IDNO:10.

Representative polypeptides and nucleic acids that are also providedcomprise orthologs from porcine and Xenopus sources, as disclosed in theExamples and in SEQ ID NOs: 80-91, and the methods, definitions,sequence comparison, and hybridization conditions set forth herein areequally applicable to the orthologs.

As disclosed further herein below, the present invention also provides asystem for functional expression of a SLC26 polypeptide, including butnot limited to a SLC26A6 polypeptide, a SLC26A1 polypeptide, and aSLC26A2 polypeptide. The system employs a recombinant SLC26 nucleicacid, including any one of odd-numbered SEQ ID NOs:1-11.

II.A. SLC26 Nucleic Acids

The terms “nucleic acid molecule” and “nucleic acid” each refer todeoxyribonucleotides or ribonucleotides and polymers thereof insingle-stranded, double-stranded, or triplexed form. Unless specificallylimited, the term encompasses nucleic acids containing known analoguesof natural nucleotides that have similar properties as the referencenatural nucleic acid. The terms “nucleic acid molecule” or “nucleicacid” can also be used in place of “gene,” “cDNA,” “mRNA,” or “cRNA.”Nucleic acids can be synthesized, or can be derived from any biologicalsource, including any organism. Representative methods for cloning afull-length SLC26 cDNA are described in Example 1.

The terms “SLC26” and terms including “SLC26” (e.g., SLC26A1, SLC26A2,and SLC26A6) are used herein to refer to nucleic acids that encode aSLC26 polypeptide. Thus, the term “SLC26” refers to isolated nucleicacids of the present invention comprising: (a) a nucleotide sequencecomprising the nucleotide sequence of any one of odd-numbered SEQ IDNOs: 1-11; or (b) a nucleotide sequence substantially identical to anyone of odd-numbered SEQ ID NOs:1-11.

The term “substantially identical”, as used herein to describe a degreeof similarity between nucleotide sequences, refers to two or moresequences that have at least about least 60%, preferably at least about70%, more preferably at least about 80%, more preferably about 90% toabout 99%, still more preferably about 95% to about 99%, and mostpreferably about 99% nucleotide identity, when compared and aligned formaximum correspondence, as measured using one of the following sequencecomparison algorithms or by visual inspection. Preferably, thesubstantial identity exists in nucleotide sequences of at least about100 residues, more preferably in nucleotide sequences of at least about150 residues, and most preferably in nucleotide sequences comprising afull length coding sequence. The term “full length” is used herein torefer to a complete open reading frame encoding a functional SLC26polypeptide, as described further herein below. Methods for determiningpercent identity between two polypeptides are defined herein below underthe heading “Nucleotide and Amino Acid Sequence Comparisons”.

In one aspect, substantially identical sequences can be polymorphicsequences. The term “polymorphic” refers to the occurrence of two ormore genetically determined alternative sequences or alleles in apopulation. An allelic difference can be as small as one base pair.

In another aspect, substantially identical sequences can comprisemutagenized sequences, including sequences comprising silent mutations.A mutation can comprise one or more residue changes, a deletion ofresidues, or an insertion of additional residues.

Another indication that two nucleotide sequences are substantiallyidentical is that the two molecules hybridize specifically to orhybridize substantially to each other under stringent conditions. In thecontext of nucleic acid hybridization, two nucleic acid sequences beingcompared can be designated a “probe” and a “target.” A “probe” is areference nucleic acid molecule, and a “target” is a test nucleic acidmolecule, often found within a heterogeneous population of nucleic acidmolecules. A “target sequence” is synonymous with a “test sequence.”

A preferred nucleotide sequence employed for hybridization studies orassays includes probe sequences that are complementary to or mimic atleast an about 14 to 40 nucleotide sequence of a nucleic acid moleculeof the present invention. Preferably, probes comprise 14 to 20nucleotides, or even longer where desired, such as 30, 40, 50, 60, 100,200, 300, or 500 nucleotides or up to the full length of any one ofodd-numbered SEQ ID NOs:1-11. Such fragments can be readily prepared by,for example, chemical synthesis of the fragment, by application ofnucleic acid amplification technology, or by introducing selectedsequences into recombinant vectors for recombinant production.

The phrase “hybridizing specifically to” refers to the binding,duplexing, or hybridizing of a molecule only to a particular nucleotidesequence under stringent conditions when that sequence is present in acomplex nucleic acid mixture (e.g., total cellular DNA or RNA).

The phrase “hybridizing substantially to” refers to complementaryhybridization between a probe nucleic acid molecule and a target nucleicacid molecule and embraces minor mismatches that can be accommodated byreducing the stringency of the hybridization media to achieve thedesired hybridization.

“Stringent hybridization conditions” and “stringent hybridization washconditions” in the context of nucleic acid hybridization experimentssuch as Southern and Northern blot analysis are both sequence- andenvironment-dependent. Longer sequences hybridize specifically at highertemperatures. An extensive guide to the hybridization of nucleic acidsis found in Tijssen (1993) Laboratory Techniques in Biochemistry andMolecular Biology-Hybridization with Nucleic Acid Probes, part I chapter2, Elsevier, New York, N.Y. Generally, highly stringent hybridizationand wash conditions are selected to be about 5° C. lower than thethermal melting point (T_(m)) for the specific sequence at a definedionic strength and pH. Typically, under “stringent conditions” a probewill hybridize specifically to its target subsequence, but to no othersequences.

The T_(m) is the temperature (under defined ionic strength and pH) atwhich 50% of the target sequence hybridizes to a perfectly matchedprobe. Very stringent conditions are selected to be equal to the T_(m)for a particular probe. An example of stringent hybridization conditionsfor Southern or Northern Blot analysis of complementary nucleic acidshaving more than about 100 complementary residues is overnighthybridization in 50% formamide with 1 mg of heparin at 42° C. An exampleof highly stringent wash conditions is 15 minutes in 0.1×SSC at 65° C.An example of stringent wash conditions is 15 minutes in 0.2×SSC bufferat 65° C. See Sambrook et al., eds (1989) Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. for a description of SSC buffer. Often, a high stringencywash is preceded by a low stringency wash to remove background probesignal. An example of medium stringency wash conditions for a duplex ofmore than about 100 nucleotides, is 15 minutes in 1×SSC at 45° C. Anexample of low stringency wash for a duplex of more than about 100nucleotides, is 15 minutes in 4× to 6×SSC at 40° C. For short probes(e.g., about 10 to 50 nucleotides), stringent conditions typicallyinvolve salt concentrations of less than about 1M Na⁺ ion, typicallyabout 0.01 to 1M Na⁺ ion concentration (or other salts) at pH 7.0-8.3,and the temperature is typically at least about 30° C. Stringentconditions can also be achieved with the addition of destabilizingagents such as formamide. In general, a signal to noise ratio of 2-fold(or higher) than that observed for an unrelated probe in the particularhybridization assay indicates detection of a specific hybridization.

The following are examples of hybridization and wash conditions that canbe used to identify nucleotide sequences that are substantiallyidentical to reference nucleotide sequences of the present invention: aprobe nucleotide sequence preferably hybridizes to a target nucleotidesequence in 7% sodium dodecyl sulphate (SDS), 0.5M NaPO₄, 1 mM EDTA at50° C. followed by washing in 2×SSC, 0.1% SDS at 50° C.; morepreferably, a probe and target sequence hybridize in 7% sodium dodecylsulphate (SDS), 0.5M NaPO₄, 1 mM EDTA at 50° C. followed by washing in1×SSC, 0.1% SDS at 50° C.; more preferably, a probe and target sequencehybridize in 7% sodium dodecyl sulphate (SDS), 0.5M NaPO₄, 1 mM EDTA at50° C. followed by washing in 0.5×SSC, 0.1% SDS at 50° C.; morepreferably, a probe and target sequence hybridize in 7% sodium dodecylsulphate (SDS), 0.5M NaPO₄, 1 mM EDTA at 50° C. followed by washing in0.1×SSC, 0.1% SDS at 50° C.; more preferably, a probe and targetsequence hybridize in 7% sodium dodecyl sulphate (SDS), 0.5M NaPO₄, 1 mMEDTA at 50° C. followed by washing in 0.1×SSC, 0.1% SDS at 65° C.

A further indication that two nucleic acid sequences are substantiallyidentical is that proteins encoded by the nucleic acids aresubstantially identical, share an overall three-dimensional structure,or are biologically functional equivalents. These terms are definedfurther under the heading “SLC26 Polypeptides” herein below. Nucleicacid molecules that do not hybridize to each other under stringentconditions are still substantially identical if the correspondingproteins are substantially identical. This can occur, for example, whentwo nucleotide sequences comprise conservatively substituted variants aspermitted by the genetic code.

The term “conservatively substituted variants” refers to nucleic acidsequences having degenerate codon substitutions wherein the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues. See Batzer et al. (1991)Nucleic Acids Res 19:5081; Ohtsuka et al. (1985) J Biol Chem260:2605-2608; and Rossolini et al. (1994) Mol Cell Probes 8:91-98.

The term “SLC26” also encompasses nucleic acids comprising subsequencesand elongated sequences of a SLC26 nucleic acid, including nucleic acidscomplementary to a SLC26 nucleic acid, SLC26 RNA molecules, and nucleicacids complementary to SLC26 RNAs (cRNAs).

The term “subsequence” refers to a sequence of nucleic acids thatcomprises a part of a longer nucleic acid sequence. An exemplarysubsequence is a probe, described herein above, or a primer. The term“primer” as used herein refers to a contiguous sequence comprising about8 or more deoxyribonucleotides or ribonucleotides, preferably 10-20nucleotides, and more preferably 20-30 nucleotides of a selected nucleicacid molecule. The primers of the invention encompass oligonucleotidesof sufficient length and appropriate sequence so as to provideinitiation of polymerization on a nucleic acid molecule of the presentinvention.

The term “elongated sequence” refers to an addition of nucleotides (orother analogous molecules) incorporated into the nucleic acid. Forexample, a polymerase (e.g., a DNA polymerase) can add sequences at the3′ terminus of the nucleic acid molecule. In addition, the nucleotidesequence can be combined with other DNA sequences, such as promoters,promoter regions, enhancers, polyadenylation signals, intronicsequences, additional restriction enzyme sites, multiple cloning sites,and other coding segments.

The term “complementary sequences,” as used herein, indicates twonucleotide sequences that comprise antiparallel nucleotide sequencescapable of pairing with one another upon formation of hydrogen bondsbetween base pairs. As used herein, the term “complementary sequences”means nucleotide sequences which are substantially complementary, as canbe assessed by the same nucleotide comparison methods set forth below,or is defined as being capable of hybridizing to the nucleic acidsegment in question under relatively stringent conditions such as thosedescribed herein. A particular example of a complementary nucleic acidsegment is an antisense oligonucleotide.

The present invention also provides chimeric genes comprising thedisclosed SLC26 nucleic acids and recombinant SLC26 nucleic acids. Thus,also included are constructs and vectors comprising SLC26 nucleic acids.

The term “gene” refers broadly to any segment of DNA associated with abiological function. A gene encompasses sequences including but notlimited to a coding sequence, a promoter region, a cis-regulatorysequence, a non-expressed DNA segment that is a specific recognitionsequence for regulatory proteins, a non-expressed DNA segment thatcontributes to gene expression, a DNA segment designed to have desiredparameters, or combinations thereof. A gene can be obtained by a varietyof methods, including cloning from a biological sample, synthesis basedon known or predicted sequence information, and recombinant derivationof an existing sequence.

The term “chimeric gene,” as used herein, refers to a promoter regionoperatively linked to a SLC26 sequence, including a SLC26 cDNA, a SLC26nucleic acid encoding an antisense RNA molecule, a SLC26 nucleic acidencoding an RNA molecule having tertiary structure (e.g., a hairpinstructure) or a SLC26 nucleic acid encoding a double-stranded RNAmolecule.

The term “operatively linked”, as used herein, refers to a functionalcombination between a promoter region and a nucleotide sequence suchthat the transcription of the nucleotide sequence is controlled andregulated by the promoter region. Techniques for operatively linking apromoter region to a nucleotide sequence are known in the art.

The term “recombinant” generally refers to an isolated nucleic acid thatis replicable in a non-native environment. Thus, a recombinant nucleicacid can comprise a non-replicable nucleic acid in combination withadditional nucleic acids, for example vector nucleic acids, which enableits replication in a host cell.

The term “vector” is used herein to refer to a nucleic acid moleculehaving nucleotide sequences that enable its replication in a host cell.A vector can also include nucleotide sequences to permit ligation ofnucleotide sequences within the vector, wherein such nucleotidesequences are also replicated in a host cell. Representative vectorsinclude plasmids, cosmids, and viral vectors. A vector can also mediaterecombinant production of a SLC26 polypeptide, as described furtherherein below.

The term “construct”, as used herein to describe a type of constructcomprising an expression construct, refers to a vector furthercomprising a nucleotide sequence operatively inserted with the vector,such that the nucleotide sequence is recombinantly expressed.

The terms “recombinantly expressed” or “recombinantly produced” are usedinterchangeably to refer generally to the process by which a polypeptideencoded by a recombinant nucleic acid is produced.

Thus, preferably, recombinant SLC26 nucleic acids comprise heterologousnucleic acids. The term “heterologous nucleic acids” refers to asequence that originates from a source foreign to an intended host cellor, if from the same source, is modified from its original form. Aheterologous nucleic acid in a host cell can comprise a nucleic acidthat is endogenous to the particular host cell but has been modified,for example by mutagenesis or by isolation from native cis-regulatorysequences. A heterologous nucleic acid also includes non-naturallyoccurring multiple copies of a native nucleotide sequence. Aheterologous nucleic acid can also comprise a nucleic acid that isincorporated into a host cell's nucleic acids at a position wherein suchnucleic acids are not ordinarily found.

Nucleic acids of the present invention can be cloned, synthesized,altered, mutagenized, or combinations thereof. Standard recombinant DNAand molecular cloning techniques used to isolate nucleic acids are knownin the art. Site-specific mutagenesis to create base pair changes,deletions, or small insertions are also known in the art. See e.g.,Sambrook et al. (eds.) (1989) Molecular Cloninq: A Laboratory Manual.Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Silhavyet al. (1984) Experiments with Gene Fusions. Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Glover & Hames (1995) DNACloning: A Practical Approach, 2nd ed. IRL Press at Oxford UniversityPress, Oxford/N.Y.; Ausubel (ed.) (1995) Short Protocols in MolecularBiology, 3rd ed. Wiley, New York.

II.B. SLC26 Polypeptides

The present invention provides novel SLC26 polypeptides comprising oneof a human SLC26A6a polypeptide, a mouse SLC26A6a polypeptide, a mouseSLC26A6b polypeptide, and a mouse SLC26A1 polypeptide. Representativeembodiments are set forth as even-numbered SEQ ID NOs:2, 6, 8, and 10,respectively. Preferably, an isolated SLC26 polypeptide of the presentinvention comprises a recombinantly expressed SLC26 polypeptide. Alsopreferably, isolated SLC26 polypeptides comprise functional SLC26polypeptides.

Thus, novel SLC26 polypeptides useful in the methods of the presentinvention comprise: (a) a polypeptide encoded by a nucleic acid of anyone of odd-numbered SEQ ID NOs:1-11; (b) a polypeptide encoded by anucleic acid substantially identical to any one of odd-numbered SEQ IDNOs:1-11; (c) a polypeptide comprising an amino acid sequence of any oneof even-numbered SEQ ID NOs:2-12; or (d) a polypeptide substantiallyidentical to any one of even-numbered SEQ ID NOs:2-12.

Representative polypeptides, and nucleic acids encoding the same, thatare provided comprise orthologs from porcine and Xenopus sources, asdisclosed in the Examples and in SEQ ID NOs: 80-91, and the definitions,sequence comparison, and hybridization conditions set forth herein areequally applicable to the orthologs.

The term “substantially identical”, as used herein to describe a levelof similarity between SLC26 and a protein substantially identical to aSLC26 protein, refers to a sequence that is at least about 35% identicalto any of even-numbered SEQ ID NOs:2-12, when compared over the fulllength of a SLC26 protein. Preferably, a protein substantially identicalto a SLC26 protein comprises an amino acid sequence that is at leastabout 35% to about 45% identical to any one of even-numbered SEQ IDNOs:2-12, more preferably at least about 45% to about 55% identical toany one of even-numbered SEQ ID NOs:2-12, even more preferably at leastabout 55% to about 65% identical to any one of even-numbered SEQ IDNOs:2-12, still more preferably preferably at least about 65% to about75% identical to any one of even-numbered SEQ ID NOs:2-12, still morepreferably preferably at least about 75% to about 85% identical to anyone of even-numbered SEQ ID NOs:2-12, still more preferably preferablyat least about 85% to about 95% identical to any one of even-numberedSEQ ID NOs:2-12, and still more preferably at least about 95% to about99% identical to any one of even-numbered SEQ ID NOs:2-12 when comparedover the full length of a SLC26 polypeptide. The term “full length”refers to a functional SLC26 polypeptide, as described further hereinbelow. Methods for determining percent identity between two polypeptidesare also defined herein below under the heading “Nucleotide and AminoAcid Sequence Comparisons”.

The term “substantially identical,” when used to describe polypeptides,also encompasses two or more polypeptides sharing a conservedthree-dimensional structure. Computational methods can be used tocompare structural representations, and structural models can begenerated and easily tuned to identify similarities around importantactive sites or ligand binding sites. See Saqi et al. (1999)Bioinformatics 15:521-522; Barton (1998) Acta Crystallogr D BiolCrystallogr 54:1139-1146; Henikoff et al. (2000) Electrophoresis21:1700-1706; and Huang et al. (2000) Pac Symp Biocomput:230-241.

Substantially identical proteins also include proteins comprising aminoacids that are functionally equivalent to amino acids of any one ofeven-numbered SEQ ID NOs:2-12. The term “functionally equivalent” in thecontext of amino acids is known in the art and is based on the relativesimilarity of the amino acid side-chain substituents. See Henikoff &Henikoff (2000) Adv Protein Chem 54:73-97. Relevant factors forconsideration include side-chain hydrophobicity, hydrophilicity, charge,and size. For example, arginine, lysine, and histidine are allpositively charged residues; that alanine, glycine, and serine are allof similar size; and that phenylalanine, tryptophan, and tyrosine allhave a generally similar shape. By this analysis, described furtherherein below, arginine, lysine, and histidine; alanine, glycine, andserine; and phenylalanine, tryptophan, and tyrosine; are defined hereinas biologically functional equivalents.

In making biologically functional equivalent amino acid substitutions,the hydropathic index of amino acids can be considered. Each amino acidhas been assigned a hydropathic index on the basis of theirhydrophobicity and charge characteristics, these are: isoleucine (+4.5);valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is generally understood inthe art (Kyte et al., 1982). It is known that certain amino acids can besubstituted for other amino acids having a similar hydropathic index orscore and still retain a similar biological activity. In making changesbased upon the hydropathic index, the substitution of amino acids whosehydropathic indices are within ±2 of the original value is preferred,those which are within ±1 of the original value are particularlypreferred, and those within ±0.5 of the original value are even moreparticularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101 describes that the greatest local average hydrophilicityof a protein, as governed by the hydrophilicity of its adjacent aminoacids, correlates with its immunogenicity and antigenicity, e.g., with abiological property of the protein. It is understood that an amino acidcan be substituted for another having a similar hydrophilicity value andstill obtain a biologically equivalent protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

In making changes based upon similar hydrophilicity values, thesubstitution of amino acids whose hydrophilicity values are within ±2 ofthe original value is preferred, those which are within ±1 of theoriginal value are particularly preferred, and those within ±0.5 of theoriginal value are even more particularly preferred.

The term “substantially identical” also encompasses polypeptides thatare biologically functional equivalents of a SLC26 polypeptide. The term“functional” includes an activity of an SLC26 polypeptide intransporting anions across a membrane. Preferably, such transport showsa magnitude and anion selectivity that is substantially similar to thatof a cognate SLC26 polypeptide in vivo. Preferably, the term“functional” also refers to similar kinetics of activation andinactivation of anion transport activity. Representative methods forassessing anion transport activity are described herein below.

The present invention also provides functional fragments of a SLC26polypeptide. Such functional portion need not comprise all orsubstantially all of the amino acid sequence of a native SLC26 geneproduct.

The present invention also includes functional polypeptide sequencesthat are longer sequences than that of a native SLC26 polypeptide. Forexample, one or more amino acids can be added to the N-terminus orC-terminus of a SLC26 polypeptide. Such additional amino acids can beemployed in a variety of applications, including but not limited topurification applications. Methods of preparing elongated proteins areknown in the art.

II.C. Nucleotide and Amino Acid Sequence Comparisons

The terms “identical” or “percent identity” in the context of two ormore nucleotide or polypeptide sequences, refer to two or more sequencesor subsequences that are the same or have a specified percentage ofamino acid residues or nucleotides that are the same, when compared andaligned for maximum correspondence, as measured using one of thesequence comparison algorithms disclosed herein or by visual inspection.

The term “substantially identical” in regards to a nucleotide orpolypeptide sequence means that a particular sequence varies from thesequence of a naturally occurring sequence by one or more deletions,substitutions, or additions, the net effect of which is to retainbiological function of a SLC26 nucleic acid or a SLC26 polypeptide.

For comparison of two or more sequences, typically one sequence acts asa reference sequence to which one or more test sequences are compared.When using a sequence comparison algorithm, test and reference sequencesare entered into a computer program, subsequence coordinates aredesignated if necessary, and sequence algorithm program parameters areselected. The sequence comparison algorithm then calculates the percentsequence identity for the designated test sequence(s) relative to thereference sequence, based on the selected program parameters.

Optimal alignment of sequences for comparison can be conducted, forexample, by the local homology algorithm of Smith & Waterman (1981) AdvAppl Math 2:482-489, by the homology alignment algorithm of Needleman &Wunsch (1970) J Mol Biol 48:443-453, by the search for similarity methodof Pearson & Lipman (1988) Proc Natl Acad Sci USA 85:2444-2448, bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, Madison, Wis.), or by visual inspection. See generally, Ausubel(ed.) (1995) Short Protocols in Molecular Biology, 3rd ed. Wiley, NewYork.

A preferred algorithm for determining percent sequence identity andsequence similarity is the BLAST algorithm, which is described inAltschul et al. (1990) J Mol Biol 215:403-410. Software for performingBLAST analyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nim.nih.gov/). This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence, which eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighborhood word score threshold. These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are then extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0) and N (penalty score for mismatching residues;always <0). For amino acid sequences, a scoring matrix is used tocalculate the cumulative score. Extension of the word hits in eachdirection are halted when the cumulative alignment score falls off bythe quantity X from its maximum achieved value, the cumulative scoregoes to zero or below due to the accumulation of one or morenegative-scoring residue alignments, or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength W=11, an expectationE=10, a cutoff of 100, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlength(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. SeeHenikoff & Henikoff (1992) Proc Natl Acad Sci USA 89:10915-10919.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences. See e.g., Karlin & Altschul (1993) Proc Natl Acad Sci USA90:5873-5877. One measure of similarity provided by the BLAST algorithmis the smallest sum probability (P(N)), which provides an indication ofthe probability by which a match between two nucleotide or amino acidsequences that would occur by chance. For example, a test nucleic acidsequence is considered similar to a reference sequence if the smallestsum probability in a comparison of the test nucleic acid sequence to thereference nucleic acid sequence is less than about 0.1, more preferablyless than about 0.01, and most preferably less than about 0.001.

III. Methods for Detecting a SLC26 Nucleic Acid

In another aspect of the invention, a method is provided for detecting anucleic acid molecule that encodes a SLC26 polypeptide. Such methods canbe used to detect SLC26 gene variants or altered gene expression. Forexample, detection of a change in SLC26 sequence or expression can beused for diagnosis of SLC26-related diseases, disorders, and druginteractions. Preferably, the nucleic acids used for this methodcomprise sequences set forth as any one of SEQ ID NOs:1, 5, 7, and 9.

Sequences detected by methods of the invention can detected, subcloned,sequenced, and further evaluated by any measure well known in the artusing any method usually applied to the detection of a specific DNAsequence. Thus, the nucleic acids of the present invention can be usedto clone genes and genomic DNA comprising the disclosed sequences.Alternatively, the nucleic acids of the present invention can be used toclone genes and genomic DNA of related sequences. Using the nucleic acidsequences disclosed herein, such methods are known to one skilled in theart. See e.g., Sambrook et al., eds (1989) Molecular Cloning, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Representativemethods are also disclosed in Examples 1-4.

In one embodiment of the invention, levels of a SLC26 nucleic acidmolecule are measured by, for example, using an RT-PCR assay. See Chiang(1998) J Chromatogr A 806:209-218, and references cited therein.

In another embodiment of the invention, genetic assays based on nucleicacid molecules of the present invention can be used to screen forgenetic variants, for example by allele-specific oligonucleotide (ASO)probe analysis (Conner et al., 1983), oligonucleotide ligation assays(OLAs) (Nickerson et al., 1990), single-strand conformation polymorphism(SSCP) analysis (Orita et al., 1989), SSCP/heteroduplex analysis, enzymemismatch cleavage, direct sequence analysis of amplified exons (Kestilaet al., 1998; Yuan et al., 1999), allele-specific hybridization(Stoneking et al., 1991), and restriction analysis of amplified genomicDNA containing the specific mutation. Automated methods can also beapplied to large-scale characterization of single nucleotidepolymorphisms (Wang et al., 1998; Brookes, 1999). Preferred detectionmethods are non-electrophoretic, including, for example, the TAQMAN™allelic discrimination assay, PCR-OLA, molecular beacons, padlockprobes, and well fluorescence. See Landegren et al. (1998) Genome Res8:769-776 and references cited therein.

IV. System for Recombinant Expression of a SLC26 Polypeptide

The present invention further provides a system for expression of arecombinant SLC26 polypeptide of the present invention. Such a systemcan be used for subsequent purification and/or characterization of aSLC26 polypeptide. For example, a purified SLC26A6 polypeptide can beused as an immunogen for the production of an SLC26 antibody, describedfurther herein below.

A system for recombinant expression of a SLC26 polypeptide can be usedfor the identification of modulators of anion transport. In oneembodiment of the invention, a method is provided for identification ofSLC26 modulators, as described herein below. Alternatively, thedisclosed SLC26 polypeptides can be used as a control anion transporterwhen testing any other molecule for anion transport activity. Forexample, the present invention discloses that SLC26A6 is a chloridetransporter, and thus a system for recombinant SLC26A6 expression can beused as a positive control in an assay to determine chloride transportof a test polypeptide. Such test polypeptides can include candidates forany one of a variety of hereditary and acquired disease such as cysticfibrosis, nephrolithiasis, and cholera.

The term “expression system” refers to a host cell comprising aheterologous nucleic acid and the polypeptide encoded by theheterologous nucleic acid. For example, a heterologous expression systemcan comprise a host cell transfected with a construct comprising arecombinant SLC26 nucleic acid, a host cell transfected with SLC26 cRNA,or a cell line produced by introduction of heterologous nucleic acidsinto a host cell genome.

A system for recombinant expression of a SLC26 polypeptide can comprise:(a) a recombinantly expressed SLC26 polypeptide; and (b) a host cellcomprising the recombinantly expressed SLC26 polypeptide. For example, aSLC26 cRNA can be transcribed in vitro and then introduced into a hostcell, whereby a SLC26 polypeptide is expressed. In a preferredembodiment of the invention, SLC26 cRNA is provided to a host cell bydirect injection of a solution comprising the SLC26 cRNA, as describedin Example 5. The system can further comprise a plurality of differentSLC26 polypeptides.

A system for recombinant expression of a SLC26 polypeptide can alsocomprise: (a) a construct comprising a vector and a nucleic acidmolecule encoding a SLC26 polypeptide operatively linked to aheterologous promoter; and (b) a host cell comprising the construct of(a), whereby the host cell expresses a SLC26 polypeptide. The system canfurther comprise constructs encoding a plurality of different SLC26polypeptides. Additionally, a single construct itself can encode aplurality of different SLC26 polypeptides.

Isolated polypeptides and recombinantly produced polypeptides can bepurified and characterized using a variety of standard techniques thatare known to the skilled artisan. See e.g., Schröder & Lübke (1965) ThePeptides. Academic Press, New York; Schneider & Eberle (1993) Peptides,1992: Proceedings of the Twenty-Second European Peptide Symposium,September 13-19, 1992, Interlaken. Switzerland. Escom, Leiden; Bodanszky(1993) Principles of Peptide Synthesis, 2nd rev. ed. Springer-Verlag,Berlin; N.Y.; Ausubel (ed.) (1995) Short Protocols in Molecular Biology,3rd ed. Wiley, New York.

Preferably, a recombinantly expressed SLC26 polypeptide comprises afunctional anion transporter. Thus, a recombinantly expressed SLC26polypeptide preferably displays transport of Cl⁻, SO₄ ²⁻, oxalate,and/or formate across a lipid bilayer or membrane. Also preferably, arecombinant SLC26 polypeptide shows ion selectivity similar to a nativeSLC26 polypeptide. Representative methods for determining SLC26 functionare described herein below.

IV.A. Expression Constructs

A construct for expression of a SLC26 polypeptide includes a vector anda SLC26 nucleotide sequence, wherein the SLC26 nucleotide sequence isoperatively linked to a promoter sequence. A construct for recombinantSLC26 expression can also comprise transcription termination signals andsequences required for proper translation of the nucleotide sequence.Preparation of an expression construct, including addition oftranslation and termination signal sequences, is known to one skilled inthe art.

Recombinant production of a SLC26 polypeptide can be directed using aconstitutive promoter or an inducible promoter. Representative promotersthat can be used in accordance with the present invention include Simianvirus 40 early promoter, a long terminal repeat promoter fromretrovirus, an actin promoter, a heat shock promoter, and a metallothienprotein.

Suitable vectors that can be used to express a SLC26 polypeptide includebut are not limited to viruses such as vaccinia virus or adenovirus,baculovirus vectors, yeast vectors, bacteriophage vectors (e.g., lambdaphage), plasmid and cosmid DNA vectors, transposon-mediatedtransformation vectors, and derivatives thereof.

Constructs are introduced into a host cell using a transfection methodcompatible with the vector employed. Standard transfection methodsinclude electroporation, DEAE-Dextran transfection, calcium phosphateprecipitation, liposome-mediated transfection, transposon-mediatedtransformation, infection using a retrovirus, particle-mediated genetransfer, hyper-velocity gene transfer, and combinations thereof.

IV.B. Host Cells

The term “host cell”, as used herein, refers to a cell into which aheterologous nucleic acid molecule can be introduced. Any suitable hostcell can be used, including but not limited to eukaryotic hosts such asmammalian cells (e.g., HeLa cells, CV-1 cells, COS cells), amphibiancells (e.g., Xenopus oocytes), insect cells (e.g., Sf9 cells), as wellas prokaryotic hosts such as E. coli and Bacillus subtilis. Preferredhost cells are amphibian cells such as Xenopus oocytes. Also preferably,a host cell substantially lacks a SLC26 polypeptide.

A host cell strain can be chosen which modulates the expression of therecombinant sequence, or modifies and processes the gene product in thespecific fashion desired. For example, different host cells havecharacteristic and specific mechanisms for the translational andpost-transactional processing and modification (e.g., glycosylation,phosphorylation of proteins). Appropriate cell lines or host systems canbe chosen to ensure the desired modification and processing of theforeign protein expressed. For example, expression in a bacterial systemcan be used to produce a non-glycosylated core protein product, andexpression in yeast will produce a glycosylated product.

The present invention further encompasses recombinant expression of aSLC26 polypeptide in a stable cell line. Methods for generating a stablecell line following transformation of a heterologous construct into ahost cell are known in the art. See e.g., Joyner (1993) Gene Targeting:A Practical Approach. Oxford University Press, Oxford/N.Y. Thus,transformed cells, tissues, or non-human organisms are understood toencompass not only the end product of a transformation process, but alsotransgenic progeny or propagated forms thereof.

The present invention further encompasses cryopreservation of cellsexpressing a recombinant SLC26 polypeptide as disclosed herein. Thus,transiently transfected cells and cells of a stable cell line expressingSLC26 can be frozen and stored for later use. Frozen cells can bereadily transported for use at a remote location.

Cryopreservation media generally consists of a base medium,cryopreservative, and a protein source. The cryopreservative and proteinprotect the cells from the stress of the freeze-thaw process. Forserum-containing medium, a typical cryopreservation medium is preparedas complete medium containing 10% glycerol; complete medium containing10% DMSO (dimethylsulfoxide), or 50% cell-conditioned medium with 50%fresh medium with 10% glycerol or 10% DMSO. For serum-free medium,typical cryopreservation formulations include 50% cell-conditioned serumfree medium with 50% fresh serum-free medium containing 7.5% DMSO; orfresh serum-free medium containing 7.5% DMSO and 10% cell culture gradeDMSO. Preferably, a cell suspension comprising about 10⁶ to about 10⁷cells per ml is mixed with cryopreservation medium.

Cells are combined with cryopreservation medium in a vial or othercontainer suitable for frozen storage, for example NUNC® CRYOTUBES™(available from Applied Scientific of South San Francisco, Calif.).Cells can also be aliquotted to wells of a multi-well plate, for examplea 96-well plate designed for high-throughput assays, and frozen inplated format.

Cells are preferably cooled from room temperature to a storagetemperature at a rate of about −1° C. per minute. The cooling rate canbe controlled, for example, by placing vials containing cells in aninsulated water-filled reservoir having about 1 liter liquid capacity,and placing such cube in a −70° C. mechanical freezer. Alternatively,the rate of cell cooling can be controlled at about −1° C. per minute bysubmersing vials in a volume of liquid refrigerant such as an aliphaticalcohol, the volume of liquid refrigerant being more than fifteen timesthe total volume of cell culture to be frozen, and placing the submersedculture vials in a conventional freezer at a temperature below about−70° C. Commercial devices for freezing cells are also available, forexample, the Planer Mini-Freezer R202/200R (Planer Products Ltd. ofGreat Britain) and the BF-5 Biological Freezer (Union CarbideCorporation of Danbury, Conn., United States of America). Preferably,frozen cells are stored at or below about −70° C. to about −80° C., andmore preferably at or below about −130° C.

To obtain the best possible cell survival, thawing of the cells must beperformed as quickly as possible. Once a vial or other reservoircontaining frozen cells is removed from storage, it should be placeddirectly into a 37° C. water bath and gently shaken until it iscompletely thawed. If cells are particularly sensitive tocryopreservatives, the cells are centrifuged to remove cryopreservativeprior to further growth.

Additional methods for preparation and handling of frozen cells can befound in Freshney (1987) Culture of Animal Cells: A Manual of BasicTechnique, 2nd ed. A. R. Liss, New York and in U.S. Pat. Nos. 6,176,089;6,140,123; 5,629,145; and 4,455,842; among other places.

V. Transgenic Animals

The present invention also provides a transgenic animal comprising adisruption of SLC26A6, SLC26A1, or SLC26A2 gene expression. Altered geneexpression can include expression of an altered level or mutated variantof a SLC26A6, SLC26A 1, or SLC26A2 gene. The present invention providesnucleic acids encoding SLC26A6, SLC26A1, and SLC26A2 that can be used toprepare constructs for generating a transgenic animal. Also provided isgenomic localization data useful for preparation of constructs targetedto the SLC26A6, SLC26A 1, or SLC26A2 locus.

In one embodiment of the present invention, the transgenic animal cancomprise a mouse with targeted modification of the mouse SLC26A6,SLC26A1, or SLC26A2 locus and can further comprise mice strains withcomplete or partial functional inactivation of the SLC26A6, SLC26A1, orSLC26A2 genes in all somatic cells.

In an alternative embodiment, a transgenic animal in accordance with thepresent invention is prepared using anti-sense or ribozyme SLC26A6,SLC26A1, or SLC26A2 constructs, driven by a universal or tissue-specificpromoter, to reduce levels of SLC26 gene expression in somatic cells,thus achieving a “knock-down” phenotype. The present invention alsoprovides the generation of murine strains with conditional or inducibleinactivation of SLC26A6, SLC26A1, SLC26A2, or a combination thereof.Such murine strains can also comprise additional synthetic or naturallyoccurring mutations, for example a mutation in any other SLC26 gene.

The present invention also provides mice strains with specific“knocked-in” modifications in the SLC26A6, SLC26A 1, or SLC26A2 genes,for example to create an over-expression or dominant negative phenotype.Thus, “knocked-in” modifications include the expression of both wildtype and mutated forms of a nucleic acid encoding a SLC26A6, SLC26A 1,or SLC26A2 polypeptide.

Techniques for the preparation of transgenic animals are known in theart. Exemplary techniques are described in U.S. Pat. No. 5,489,742(transgenic rats); U.S. Pat. Nos. 4,736,866, 5,550,316, 5,614,396,5,625,125 and 5,648,061 (transgenic mice); U.S. Pat. No. 5,573,933(transgenic pigs); U.S. Pat. No. 5,162,215 (transgenic avian species)and U.S. Pat. No. 5,741,957 (transgenic bovine species), the entirecontents of each of which are herein incorporated by reference.

For example, a transgenic animal of the present invention can comprisesa mouse with targeted modification of the mouse SLC26A6, SLC26A 1, orSLC26A2 gene. Mice strains with complete or partial functionalinactivation of the SLC26A6, SLC26A 1, or SLC26A2 genes in all somaticcells are generated using standard techniques of site-specificrecombination in murine embryonic stem cells. See Capecchi, M. R. (1989)Science 244(4910):1288-92; Thomas, K. R., and Capecchi, M. R. (1990)Nature 346(6287):847-50; Delpire, E., et al. (1999) Nat Genet22(2):192-5.

VI. SLC26 Antibodies

In another aspect of the invention, a method is provided for producingan antibody that specifically binds a SLC26 polypeptide. According tothe method, a full-length recombinant SLC26 polypeptide, or fragmentthereof, is formulated so that it can be used as an effective immunogen,and used to immunize an animal so as to generate an immune response inthe animal. The immune response is characterized by the production ofantibodies that can be collected from the blood serum of the animal. Thepresent invention also provides antibodies produced by methods thatemploy the novel SLC26 polypeptides disclosed herein, including any oneof SEQ ID NOs:2, 6, 8, and 10.

The term “antibody” refers to an immunoglobulin protein, or functionalportion thereof, including a polyclonal antibody, a monoclonal antibody,a chimeric antibody, a hybrid antibody, a single chain antibody, amutagenized antibody, a humanized antibody, and antibody fragments thatcomprise an antigen binding site (e.g., Fab and Fv antibody fragments).In a preferred embodiment of the invention, a SLC26 antibody comprises amonoclonal antibody. Thus, the present invention also encompassesantibodies and cell lines that produce monoclonal antibodies asdescribed herein.

The term “specifically binds”, when used to describe binding of anantibody to a SLC26 polypeptide, refers to binding to a SLC26polypeptide in a heterogeneous mixture of other polypeptides.

The phrases “substantially lack binding” or “substantially no binding”,as used herein to describe binding of an antibody to a controlpolypeptide or sample, refers to a level of binding that encompassesnon-specific or background binding, but does not include specificbinding.

Techniques for preparing and characterizing antibodies are known in theart. See e.g., Harlow & Lane (1988) Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and U.S.Pat. Nos. 4,196,265; 4,946,778; 5,091,513; 5,132,405; 5,260,203;5,677,427; 5,892,019; 5,985,279; 6,054561.

SLC26 antibodies prepared as disclosed herein can be used in methodsknown in the art relating to the localization and activity of SLC26polypeptides, e.g., for cloning of nucleic acids encoding a SLC26polypeptide, immunopurification of a SLC26 polypeptide, imaging a SLC26polypeptide in a biological sample, and measuring levels of a SLC26polypeptide in appropriate biological samples. To perform such methods,an antibody of the present invention can further comprise a detectablelabel, including but not limited to a radioactive label, a fluorescentlabel, an epitope label, and a label that can be detected in vivo.Methods for selection of a label suitable for a particular detectiontechnique, and methods for conjugating to or otherwise associating adetectable label with an antibody are known to one skilled in the art.

VII. SLC26 Modulators

The present invention further discloses assays to identify modulators ofSLC26 activity. An assay can employ a system for expression of a SLC26polypeptide, as disclosed herein above, or an isolated SLC26 polypeptideproduced in such a system. The present invention also providesmodulators of anion transport activity identified using the disclosedmethods.

The term “modulate” means an increase, decrease, or other alteration ofany or all chemical and biological activities or properties of a SLC26polypeptide. Thus, the method for identifying modulators involvesassaying a level or quality of SLC26 function.

A method for identifying a modulator of anion transport can comprise:(a) providing a recombinant expression system whereby a SLC26polypeptide is expressed in a host cell, and wherein the SLC26polypeptide comprises a human SLC26A6a polypeptide, a mouse SLC26A6polypeptide, or a mouse SLC26A1 polypeptide; (b) providing a testsubstance to the system of (a); (c) assaying a level or quality of SLC26function in the presence of the test substance; (d) comparing the levelor quality of SLC26 function in the presence of the test substance witha control level or quality of SLC26 function; and (e) identifying a testsubstance as an anion transport modulator by determining a level orquality of SLC26 function in the presence of the test substance assignificantly changed when compared to a control level or quality ofSLC26 function.

In one embodiment of the invention, assaying SLC26 function comprisesdetermining a level of SLC26 expression.

In another embodiment of the invention, assaying SLC26 functioncomprises assaying binding activity of a recombinantly expressed SLC26polypeptide. For example, a SLC26 activity can comprise an amount or astrength of binding of a modulator to a SLC26 polypeptide.

In still another embodiment of the invention, assaying SLC26 functioncan comprise assaying an active conformation of a SLC26 polypeptide.

In a preferred embodiment of the invention, assaying SLC26 activitycomprises assaying anion transport activity of a recombinantly expressedSLC26 polypeptide. A representative level of SLC26 activity can thuscomprise an amount of anion transport or a peak level of aniontransport, measurable as described in Example 6. A representativequality of SLC26 activity can comprise, for example, anion selectivityof a SLC26A, pH sensitivity of anion transport, and pharmacologicalsensitivity of a SLC26 polypeptide. The electrophysiological behavior ofSLC26A6 and other SLC26 polypeptides also provides a signature fortransport activity.

A control level or quality of SLC26 activity refers to a level orquality of wild type SLC26 activity. Preferably, a system forrecombinant expression of a SLC26 polypeptide comprises any one ofeven-numbered SEQ ID NOs:2-12. When evaluating the modulating capacityof a test substance, a control level or quality of SLC26 activitycomprises a level or quality of activity in the absence of a testsubstance.

The term “significantly changed”, as used herein to refer to an alteredlevel or activity of a SLC26 polypeptide, refers to a quantified changein a measurable quality that is larger than the margin of error inherentin the measurement technique, preferably an increase or decrease byabout 2-fold or greater relative to a control measurement, morepreferably an increase or decrease by about 5-fold or greater, and mostpreferably an increase or decrease by about 10-fold or greater.

Modulators identified by the disclosed methods can comprise agonists andantagonists. As used herein, the term “agonist” means a substance thatactivates, synergizes, or potentiates the biological activity of a SLC26polypeptide. As used herein, the term “antagonist” refers to a substancethat blocks or mitigates the biological activity of a SLC26 polypeptide.A modulator can also comprise a ligand or a substance that specificallybinds to a SLC26 polypeptide. Activity and binding assays for thedetermination of a SLC26 modulator can be performed in vitro or in vivo.

In one embodiment of the invention, such assays are useful for theidentification of SLC26 modulators that can be developed for thetreatment and/or diagnosis of SLC26-related disorders, as describedfurther herein below under the heading “Therapeutic Applications.”

In another embodiment of the invention, assays using a recombinant SLC26polypeptide can be performed for the purpose of prescreening bioactiveagents, wherein an interaction between the agent and SLC26 isundesirable. Thus, drugs intended for administration to a subject forthe treatment of a non-SLC26-related disorder can be tested for SLC26modulating activity that can result in undesirable side effects. Thedisclosed assays and methods enable pre-screening of bioactive agentsunder development to identify deleterious effects of anion transport.

In still another embodiment of the invention, an assay disclosed hereincan be used to characterize a mutant SLC26 polypeptide, for example amutant polypeptide that is linked to a disorder of anion transport.Recombinant expression of mutated SLC26 polypeptides will permit furtheranalysis of disorder-related SLC26 anion transporters.

In accordance with the present invention there is also provided a rapidand high throughput screening method that relies on the methodsdescribed herein. This screening method comprises separately contactinga SLC26 polypeptide with a plurality of test substances. In such ascreening method the plurality of target substances preferably comprisesmore than about 10⁴ samples, or more preferably comprises more thanabout 10⁵ samples, and still more preferably more than about 10⁶samples.

VII.A. Test Substances

A potential modulator assayed using the methods of the present inventioncomprises a candidate substance. As used herein, the terms “candidatesubstance” and “test substance” are used interchangeably, and eachrefers to a substance that is suspected to interact with a SLC26polypeptide, including any synthetic, recombinant, or natural product orcomposition. A test substance suspected to interact with a polypeptidecan be evaluated for such an interaction using the methods disclosedherein.

Representative test substances include but are not limited to peptides,oligomers, nucleic acids (e.g., aptamers), small molecules (e.g.,chemical compounds), antibodies or fragments thereof, nucleicacid-protein fusions, any other affinity agent, and combinationsthereof. A test substance can additionally comprise a carbohydrate, avitamin or derivative thereof, a hormone, a neurotransmitter, a virus orreceptor binding domain thereof, an opsin or rhodopsin, an odorant, aphermone, a toxin, a growth factor, a platelet activation factor, aneuroactive peptide, or a neurohormone. A candidate substance to betested can be a purified molecule, a homogenous sample, or a mixture ofmolecules or compounds.

The term “small molecule” as used herein refers to a compound, forexample an organic compound, with a molecular weight of less than about1,000 daltons, more preferably less than about 750 daltons, still morepreferably less than about 600 daltons, and still more preferably lessthan about 500 daltons. A small molecule also preferably has a computedlog octanol-water partition coefficient in the range of about −4 toabout +14, more preferably in the range of about −2 to about +7.5.

Test substances can be obtained or prepared as a library. As usedherein, the term “library” means a collection of molecules. A librarycan contain a few or a large number of different molecules, varying fromabout ten molecules to several billion molecules or more. A molecule cancomprise a naturally occurring molecule, a recombinant molecule, or asynthetic molecule. A plurality of test substances in a library can beassayed simultaneously. Optionally, test substances derived fromdifferent libraries can be pooled for simultaneous evaluation.

Representative libraries include but are not limited to a peptidelibrary (U.S. Pat. Nos. 6,156,511, 6,107,059, 5,922,545, and 5,223,409),an oligomer library (U.S. Pat. Nos. 5,650,489 and 5,858,670), an aptamerlibrary (U.S. Pat. Nos. 6,180,348 and 5,756,291), a small moleculelibrary (U.S. Pat. Nos. 6,168,912 and 5,738,996), a library ofantibodies or antibody fragments (U.S. Pat. Nos. 6,174,708, 6,057,098,5,922,254, 5,840,479, 5,780,225, 5,702,892, and 5,667,988), a library ofnucleic acid-protein fusions (U.S. Pat. No. 6,214,553), and a library ofany other affinity agent that can potentially bind to a SLC26polypeptide (e.g., U.S. Pat. Nos. 5,948,635, 5,747,334, and 5,498,538).

A library can comprise a random collection of molecules. Alternatively,a library can comprise a collection of molecules having a bias for aparticular sequence, structure, or conformation. See e.g., U.S. Pat.Nos. 5,264,563 and 5,824,483. Methods for preparing libraries containingdiverse populations of various types of molecules are known in the art,for example as described in U.S. Patents cited herein above. Numerouslibraries are also commercially available.

VII.B. Expression Assays

The present invention also provides a method for identifying a substancethat regulates SLCA26A6 gene expression. The term “gene expression” isused herein to refer generally to the cellular processes by which afunctional SLC26 polypeptide is produced from a nucleic acid. Thus, aSLC26 modulator can comprise a substance that binds to and regulates aSLC26A6 promoter.

The term “promoter” refers to a nucleic acid that can direct geneexpression of a nucleic acid to which it is operatively linked. Arepresentative SLC26A6 promoter is set forth as SEQ ID NO:13.

Reporter Gene Assay. In one embodiment of the invention, a geneexpression assay utilizes a chimeric gene that includes an isolatedSLCA26A6 promoter region operably linked to a reporter gene. Accordingto this method, a gene expression system is established that includesthe chimeric gene and components required for gene transcription andtranslation so that reporter gene expression is assayable. To select asubstance that modulates SLCA26A6 expression, the method furtherprovides the steps of using the gene expression system to determine abaseline level of reporter gene expression in the absence of a testsubstance, providing a plurality of test substances to the geneexpression system, and assaying a level of reporter gene expression inthe presence of a test substance. A test substance is selected whosepresence results in an altered level of reporter gene expression whencompared to the baseline level.

To perform the disclosed method, the present invention further providesa chimeric gene comprising a SLCA26A6 promoter region operably linked toa heterologous nucleotide sequence. Preferably, the SLCA26A6 promoterregion comprises the nucleic acid molecule of SEQ ID NO:13, orfunctional portion thereof. In a preferred embodiment, a chimeric geneof the invention is carried in a vector and expressed in a host cell.Preferred host cells include mammalian cells, for example HeLa cells.

The terms “reporter gene,” “marker gene,” and “selectable marker” eachrefer to a heterologous gene encoding a product that is readily observedand/or quantitated. Non-limiting examples of detectable reporter genesthat can be operatively linked to a transcriptional regulatory regioncan be found in Alam & Cook (1990) Anal Biochem 188:245-254 and in PCTInternational Publication No. WO 97/47763. Preferred reporter genes fortranscriptional analyses include the lacZ gene (Rose & Botstein, 1983),Green Fluorescent Protein (GFP) (Cubitt et al., 1995), luciferase, orchloramphenicol acetyl transferase (CAT).

An amount of reporter gene can be assayed by any method forqualitatively or preferably, quantitatively determining presence oractivity of the reporter gene product. The amount of reporter geneexpression directed by each test promoter region fragment is compared toan amount of reporter gene expression to a control construct comprisingthe reporter gene in the absence of a promoter region fragment. Apromoter region fragment is identified as having promoter activity whenthere is significant increase in an amount of reporter gene expressionin a test construct as compared to a control construct.

Representative methods for reporter gene assays can be found in U.S.Pat. No. 6,087,111, among other places.

One-Hybrid Analysis. Modulators that bind a SLC26A6 promoter can also beidentified using one-hybrid analysis. According to this approach, aSLC26A6 promoter is operatively linked to one, or typically more, yeastreporter genes such as the lacZ gene, the URA3 gene, the LEU2 gene, theHIS3 gene, or the LYS2 gene, and the reporter gene fusion construct(s)is inserted into an appropriate yeast host strain. It is expected thatthe reporter genes are not transcriptionally active in the engineeredyeast host strain, for lack of a transcriptional activator protein tobind the SLC26A6 promoter. The engineered yeast host strain istransformed with a library of cDNAs inserted in a yeast activationdomain fusion protein expression vector, e.g. pGAD, where the codingregions of the cDNA inserts are fused to a functional yeast activationdomain coding segment, such as those derived from the GAL4 or VP16activators. Transformed yeast cells that acquire a cDNA encoding aprotein that binds a cis-regulatory element of a SLC26A6 promoter can beidentified based on the concerted activation the reporter genes, eitherby genetic selection for prototrophy (e.g., LEU2, HIS3, or LYS2reporters) or by screening with chromogenic substrates (lacZ reportergene) by methods known in the art. See e.g., Luo et al. (1996)Biotechniques 20:564-568; Vidal et al. (1996) Proc Natl Acad Sci USA93:10315-10320; and Li & Herskowitz (1993) Science 262:1870-1874.

In Situ Filter Detection. About 10⁷ λgt11 clones of a cDNA expressionlibrary are prepared poly(A)⁺ RNA derived from a tissue where SLC26A6 isnormally expressed (e.g., kidney). Clones are plated and replicated onnitrocellulose filters. After denaturation and renaturation, thefilter-bound proteins are screened with a concatenated oligonucleotideprobe containing the nucleotide sequence of a SLC26A6 promoter. Theprobe is prepared by nick translation with a specific activity of>10⁸/mg. Duplicate screening using a probe carrying a mutated SLC26A6promoter is carried out to eliminate false positive clones.

VII.C. Binding Assays

In another embodiment, a method for identifying of a SLC26 modulatorcomprises determining specific binding of a test substance to a SLC26polypeptide. The term “binding” refers to an affinity between twomolecules. Preferably, specific binding also encompasses a quality orstate of mutual action such that an activity of one protein or compoundon another protein is inhibitory (in the case of an antagonist) orenhancing (in the case of an agonist).

The phrase “specifically (or selectively) binds”, when referring to thebinding capacity of a candidate modulator, refers to a binding reactionwhich is determinative of the presence of the protein in a heterogeneouspopulation of proteins and other biological materials. The binding of amodulator to a SLC26 polypeptide can be considered specific if thebinding affinity is about 1×10⁴M⁻¹ to about 1×10⁶M⁻¹ or greater. Thephrase “specifically binds” also refers to saturable binding. Todemonstrate saturable binding of a test substance to a SLC26polypeptide, Scatchard analysis can be carried out as described, forexample, by Mak et al. (1989) J Biol Chem 264:21613-21618.

The phases “substantially lack binding” or “substantially no binding”,as used herein to describe binding of a modulator to a controlpolypeptide or sample, refers to a level of binding that encompassesnon-specific or background binding, but does not include specificbinding.

Several techniques can be used to detect interactions between a SLC26polypeptide and a test substance without employing a known competitivemodulator. Representative methods include, but are not limited to,Fluorescence Correlation Spectroscopy, Surface-Enhanced LaserDesorption/Ionization Time-Of-flight Spectroscopy, and Biacoretechnology, each technique described herein below. These methods areamenable to automated, high-throughput screening.

Fluorescence Correlation Spectroscopy. Fluorescence CorrelationSpectroscopy (FCS) measures the average diffusion rate of a fluorescentmolecule within a small sample volume (Tallgren, 1980). The sample sizecan be as low as 10³ fluorescent molecules and the sample volume as lowas the cytoplasm of a single bacterium. The diffusion rate is a functionof the mass of the molecule and decreases as the mass increases. FCS cantherefore be applied to polypeptide-ligand interaction analysis bymeasuring the change in mass and therefore in diffusion rate of amolecule upon binding. In a typical experiment, the target to beanalyzed (e.g., a SLC26 polypeptide) is expressed as a recombinantpolypeptide with a sequence tag, such as a poly-histidine sequence,inserted at the N-terminus or C-terminus. The expression is mediated ina host cell, such as E. coli, yeast, Xenopus oocytes, or mammaliancells. The polypeptide is purified using chromatographic methods. Forexample, the poly-histidine tag can be used to bind the expressedpolypeptide to a metal chelate column such as Ni²⁺ chelated oniminodiacetic acid agarose. The polypeptide is then labeled with afluorescent tag such as carboxytetramethylrhodamine or BODIPY™ reagent(available from Molecular Probes of Eugene, Oreg.). The polypeptide isthen exposed in solution to the potential ligand, and its diffusion rateis determined by FCS using instrumentation available from Carl Zeiss,Inc. (Thornwood, N.Y.). Ligand binding is determined by changes in thediffusion rate of the polypeptide.

Surface-Enhanced Laser DesorDtion/lonization. Surface-Enhanced LaserDesorption/lonization (SELDI) was developed by Hutchens & Yip (1993)Rapid Commun Mass Spectrom 7:576-580. When coupled to a time-of-flightmass spectrometer (TOF), SELDI provides a technique to rapidly analyzemolecules retained on a chip. It can be applied to ligand-proteininteraction analysis by covalently binding the target protein, orportion thereof, on the chip and analyzing by mass spectrometry thesmall molecules that bind to this protein (Worrall et al., 1998). In atypical experiment, a target polypeptide (e.g., a SLC26 polypeptide) isrecombinantly expressed and purified. The target polypeptide is bound toa SELDI chip either by utilizing a poly-histidine tag or by otherinteraction such as ion exchange or hydrophobic interaction. A chip thusprepared is then exposed to the potential ligand via, for example, adelivery system able to pipet the ligands in a sequential manner(autosampler). The chip is then washed in solutions of increasingstringency, for example a series of washes with buffer solutionscontaining an increasing ionic strength. After each wash, the boundmaterial is analyzed by submitting the chip to SELDI-TOF. Ligands thatspecifically bind a target polypeptide are identified by the stringencyof the wash needed to elute them.

Biacore. Biacore relies on changes in the refractive index at thesurface layer upon binding of a ligand to a target polypeptide (e.g., aSLC26 polypeptide) immobilized on the layer. In this system, acollection of small ligands is injected sequentially in a 2-5 microlitercell, wherein the target polypeptide is immobilized within the cell.Binding is detected by surface plasmon resonance (SPR) by recordinglaser light refracting from the surface. In general, the refractiveindex change for a given change of mass concentration at the surfacelayer is practically the same for all proteins and peptides, allowing asingle method to be applicable for any protein (Liedberg et al., 1983;Malmquist, 1993). In a typical experiment, a target protein isrecombinantly expressed, purified, and bound to a Biacore chip. Bindingcan be facilitated by utilizing a poly-histidine tag or by otherinteraction such as ion exchange or hydrophobic interaction. A chip thusprepared is then exposed to one or more potential ligands via thedelivery system incorporated in the instruments sold by Biacore(Uppsala, Sweden) to pipet the ligands in a sequential manner(autosampler). The SPR signal on the chip is recorded and changes in therefractive index indicate an interaction between the immobilized targetand the ligand. Analysis of the signal kinetics of on rate and off rateallows the discrimination between non-specific and specific interaction.See also Homola et al. (1999) Sensors and Actuators 54:3-15 andreferences therein.

VII.D. Conformational Assay

The present invention also provides a method for identifying a SLC26modulator that relies on a conformational change of a SLC26 polypeptidewhen bound by or otherwise interacting with a SLC26 modulator.

Application of circular dichroism to solutions of macromolecules revealsthe conformational states of these macromolecules. The technique candistinguish random coil, alpha helix, and beta chain conformationalstates.

To identify modulators of SLC26A, circular dichroism analysis can beperformed using recombinantly expressed SLC26A. A SLC26 polypeptide ispurified, for example by ion exchange and size exclusion chromatography,and mixed with a test substance. The mixture is subjected to circulardichroism. The conformation of a SLC26 polypeptide in the presence of atest substance is compared to a conformation of a SLC26 polypeptide inthe absence of a test substance. A change in conformational state of aSLC26 polypeptide in the presence of a test substance can thus be usedto identify a SLC26 modulator. Representative methods are described inU.S. Pat. Nos. 5,776,859 and 5,780,242.

VII.E. Anion Transport Assays

In a preferred embodiment of the invention, a method for identifying aSLC26 modulator employs a functional SLC26 polypeptide. Novel functionalSLC26 polypeptides disclosed herein include any of SEQ ID NOs:2, 6, 8,and 10. Representative methods for determining anion transport activityof a functional SLC26 modulator include measuring anion flux anddetermining electrogenic transport, each described briefly herein below.

In accordance with the method, cells expressing SLC26 can be provided inthe form of a kit useful for performing an assay of SLC26 function.Thus, cells can be frozen as described herein above and transportedwhile frozen to others for performance of an assay. For example, in oneembodiment of the invention, a test kit is provided for detecting aSLC26 modulator, the kit comprising: (a) frozen cells transfected withDNA encoding a full-length SLC26 polypeptide; and (b) a medium forgrowing the cells.

Preferably, a cell used in such an assay comprises a cell that issubstantially devoid of native SLC26 and polypeptides substantiallysimilar to SLC26. A preferred cell comprises a vertebrate cell, forexample a Xenopus oocyte. In one embodiment of the invention, a cellused in the assay comprises a stable cell line that recombinantlyexpresses SLC26. Alternatively, a cell used in the assay can transientlyexpress a SLC26 polypeptide as described in Example 5.

The term “substantially devoid of”, as used herein to describe a hostcell or a control cell, refers to a quality of having a level of nativeSLC26A, a level of a polypeptide substantially similar to SLC26A, or alevel of activity thereof, comprising a background level. The term“background level” encompasses non-specific measurements of expressionor activity that are typically detected in a cell free of SLC26 and freeof polypeptides substantially similar to SLC26A.

Also preferably, all assays employing cells expressing recombinant SLC26additionally employ control cells that are substantially devoid ofnative SLC26 and polypeptides substantially similar to SLC26A. Whenusing transiently transfected cells, a control cell can comprise, forexample, an untransfected host cell. When using a stable cell lineexpressing SLC26A, a control cell can comprise, for example, a parentcell line used to derive the SLC26A-expressing cell line.

Assays of SLC26 activity that employ transiently transfected cellspreferably include a marker that distinguishes transfected cells fromnon-transfected cells. The term “marker” refers to any detectablemolecule that can be used to distinguish a cell that recombinantlyexpresses SLC26 from a cell that does not recombinantly express a SLC26polypeptide. Preferably, a marker is encoded by or otherwise associatedwith a construct for SLC26 expression, such that cells aresimultaneously transfected with a nucleic acid molecule encoding SLC26and the marker. Representative detectable molecules that are useful asmarkers include but are not limited to a heterologous nucleic acid, apolypeptide encoded by a transfected construct (e.g., an enzyme or afluorescent polypeptide), a binding protein, and an antigen.

A marker comprising a heterologous nucleic acid includes nucleic acidsencoding a SLC26 polypeptide. Alternatively, any suitable method can beused to detect the encoded SLC26 polypeptide, as described herein below.

Examples of enzymes that are useful as markers include phosphatases(such as acid or alkaline phosphatase), β-galactosidase, urease, glucoseoxidase, carbonic anhydrase, acetylcholinesterase, glucoamylase, maleatedehydrogenase, glucose-6-phosphate dehydrogenase, β-glucosidase,proteases, pyruvate decarboxylase, esterases, luciferase, alcoholdehydrogenase, or peroxidases (such as horseradish peroxidase).

A marker comprising an enzyme can be detected based on activity of theenzyme. Thus, a substrate is be added to catalyze a reaction the endproduct of which is detectable, for example using spectrophotometer, aluminometer, or a fluorimeter. Substrates for reaction by theabove-mentioned enzymes, and that produce a detectable reaction product,are known to one of skill in the art.

A preferred marker comprises an encoded polypeptide that can be detectedin the absence of an added substrate. Representative polypeptides thatcan be detected directly include GFP and EGFP. Common research equipmenthas been developed to perform high-throughput detection of fluorescence,for example GFP or EGFP fluorescence, including instruments from GSILumonics (Watertown, Mass., United States of America), AmershamPharmacia Biotech/Molecular Dynamics (Sunnyvale, Calif., United Statesof America), Applied Precision Inc. (Issauah, Wash., United States ofAmerica), and Genomic Solutions Inc. (Ann Arbor, Mich., United States ofAmerica). Most of the commercial systems use some form of scanningtechnology with photomultiplier tube detection.

Anion Flux Assay. A candidate substance can be tested for its ability tomodulate a SLC26 polypeptide by determining anion flux across a membraneor lipid bilayer. Anion levels can be determined by any suitableapproach. For example, an anion can be detected using a radiolabeledanion as described in Example 6.

Anion flux can also be measured using any of a variety of indicatorcompounds. Preferably, an indicator compound comprises a compound thatcan be detected in a high-throughput capacity. Representativefluorescent indicators useful for detecting halides (e.g., chloride)include quinolium-type Cl⁻ indicators (Verkman, 1990; Mansoura et al.,1999), cell-permeable indicators (Biwersi & Verkman, 1991), ratiometricindicators (Biwersi & Verkman, 1991), and long wavelength indicators(Biwersi et al., 1994; Jayaraman et al., 1999). An indicator can alsocomprise a recombinant protein. For example, the yellow fluorescentprotein mutant, YFP-H148Q, produces fluorescence that is decreased uponhalide binding (Jayaraman et al., 2000; Galietta et al., 2001). Suchindicators are compatible with high-throughput assay formats and can bedetected using, for example, an instrument for fluorescent detection asnoted herein above.

Anion flux in a population of cultured cells can also be measured basedon changes in a degree of light scattering that is correlated with cellsize. See e.g., Krick et al. (1998) Pflugers Arch 435:415-421.

An anion flux assay can also comprise a competitive assay design. Forexample, the method can comprise: (a) providing an expression system,whereby a functional SLC26 polypeptide is expressed; (b) adding a SLC26activator to the expression system, whereby anion transport is elicited;(c) adding a test substance to the expression system; and (d) observinga suppression of the anion transport in the presence of the SLC26activator and the test substance, whereby an inhibitor of SLC26 isdetermined. Optionally, the persistent activator and test substance canbe provided to the functional expression simultaneously. Similarly, anassay for determining a SLC26 activator can comprise steps (a)-(d) abovewith the exception that an enhancement of conductance is observed in thepresence of the persistent activator and the test substance.

Electrogenic Transport Assay. Anion transport via a SLC26 polypeptide ofthe present invention can further be determined to be electrogenic bymonitoring changes in intracellular pH (pH_(i)) or membrane voltage(V_(m)) during transport. Representative methods are described by Romeroet al. (1998) Am J Physiol 274:F425-432 and Romero et al. (2000) J BiolChem 275:24552-24559.

Briefly, an oocyte is visualized with a dissecting microscope and heldon a nylon mesh in a chamber having a volume of about 250 μl. The oocyteis continuously superfused with a saline solution (3 ml/min to 5 ml/min)that is delivered through TYGON® tubing (Worchester, Mass., UnitedStates of America). Solutions can be switched using a daisy-chain systemof computer-actuated five-way valves with zero dead space. Solutionchanges in the chamber typically occur within 15 seconds to about 20seconds. Membrane voltage (V_(m)) and intracellular pH (pH_(i)) of X.laevis oocytes are measured simultaneously using microelectrodes, asdescribed by Romero et al. (1997) Nature 387:409-413.

V_(m) electrodes can be pulled from borosilicate fiber-capillary glass(Warner Instruments of West Haven, Conn., United States of America).Electrodes are backfilled with 3M KCl and typically have a resistance ofabout 3MΩ to 5MΩ. The pH electrodes can be pulled in a similar manner,and are silanized by exposing them to 40 μl ofbis-di-(methylamino)-dimethylsilane (Fluka Chemical of Ronkonkoma, N.Y.,United States of America) for 5 minutes to 10 minutes. Silanizedelectrodes are deposited in an enclosed container at 200° C., and thenbaked overnight. The pH micropipettes are cooled under vacuum, and theirtips are filled with hydrogen ionophore 1-cocktail B (Fluka Chemical ofRonkonkoma, N.Y., United States of America). The pH micropipettes arethen backfilled with a buffer containing 0.04M KH₂PO₄, 0.023M NaOH, and0.015M NaCl (pH 7.0). Representative pH microelectrodes have slopesranging from about −54 mV/pH unit to −59 mV/pH unit.

The V_(m) and pH_(i) electrodes are connected to high-impedanceelectrometers as described by Davis et al. (1992) Am J Physiol263:C246-256 and Siebens & Boron (1989) Am J Physiol 256:F354-365. Thevoltage due to pH can be obtained by electronically subtracting thesignals from the pH and V_(m) electrodes. V_(m) can be obtained bysubtracting the signals from the V_(m) electrode and an externalreference (calomel) electrode.

In accordance with the methods of the present invention, electrogenictransport can be detected using any suitable method. For example, pH canalso be assayed by detecting the presence of a fluorescence dye, forexample BCECF (available from Photon Technology International, Inc. ofLawrenceville, N.J., United States of America).

Vesicle Transport Assays. Once a SLC26 modulator has been identified,its effectiveness in modulating anion transport activity can further betested in isolated membrane vesicles, including brush border membranevesicles derived from kidney and gut. Modulators can also be tested foractivity in cultured grafts, for example intact renal proximal tubules.Methods for preparing membrane vesicles and exografts are known in theart, and representative protocols are described by Pritchard & Miller(1993) Physiol Rev 73:765-796; Miller et al. (1996) Am J Physiol271:F508-520; Masereeuw et al. (1996) Am J Physiol 271:F1173-1182;Masereeuw et al. (1999) J Pharmacol Exp Ther 289:1104-1111; Hagenbuch etal. (1985) Pflugers Arch 405:202-208; Kuo & Aronson (1988) J Biol Chem263:9710-9717; and Pritchard & Renfro (1983) Proc Natl Acad Sci USA80:2603-2607.

VII.F. Rational Design

The knowledge of the structure a native SLC26 polypeptide provides anapproach for rational design of modulators and diagnostic agents. Inbrief, the structure of a SLC26 polypeptide can be determined by X-raycrystallography and/or by computational algorithms that generatethree-dimensional representations. See Saqi et al. (1999) Bioinformatics15:521-522; Huang et al. (2000) Pac Symp Biocomput:230-241; and PCTInternational Publication No. WO 99/26966. Alternatively, a workingmodel of a SLC26 polypeptide structure can be derived by homologymodeling (Maalouf et al., 1998). Computer models can further predictbinding of a protein structure to various substrate molecules that canbe synthesized and tested using the assays described herein above.Additional compound design techniques are described in U.S. Pat. Nos.5,834,228 and 5,872,011.

In general, a SLC26 polypeptide is a membrane protein, and can bepurified in soluble form using detergents or other suitable amphiphilicmolecules. The resulting SLC26 polypeptide is in sufficient purity andconcentration for crystallization. The purified SLC26 polypeptidepreferably runs as a single band under reducing or non-reducingpolyacrylamide gel electrophoresis (PAGE). The purified SLC26polypeptide can be crystallized under varying conditions of at least oneof the following: pH, buffer type, buffer concentration, salt type,polymer type, polymer concentration, other precipitating ligands, andconcentration of purified SLC26. Methods for generating a crystallinepolypeptide are known in the art and can be reasonably adapted fordetermination of a SLC26 polypeptide as disclosed herein. See e.g.,Deisenhofer et al. (1984) J Mol Biol 180:385-398; Weiss et al. (1990)FEBS Lett 267:268-272; or the methods provided in a commercial kit, suchas the CRYSTAL SCREEN™ kit (available from Hampton Research ofRiverside, Calif., United States of America).

A crystallized SLC26 polypeptide can be tested for functional activityand differently sized and shaped crystals are further tested forsuitability in X-ray diffraction. Generally, larger crystals providebetter crystallography than smaller crystals, and thicker crystalsprovide better crystallography than thinner crystals. Preferably, SLC26crystals range in size from 0.1-1.5 mm. These crystals diffract X-raysto at least 10 Å resolution, such as 1.5-10.0 Å or any range of valuetherein, such as 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5,2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5 or 3, with 3.5 Å orless being preferred for the highest resolution.

VIII. Methods for Detecting a SLC26 Polypeptide

The present invention further provides methods for detecting a SLC26polypeptide. The disclosed methods can be used for determining alteredlevels of SLC26 expression that are associated with disorders anddisease states, including but not limited to conditions of oxalatehyperexcretion (for example, in renal stone disease), CF-related andidiopathic pancreatitis, hypertension, edema, and other conditions ofabnormal salt, oxalate, or bicarbonate transport.

In one embodiment of the invention, the method involves performing animmunochemical reaction with an antibody that specifically recognizes aSLC26 polypeptide, wherein the antibody was prepared according to amethod of the present invention for producing such an antibody. Thus,the method comprises: (a) obtaining a biological sample comprisingpeptidic material; (b) contacting the biological sample with an antibodythat specifically binds a SLC26 polypeptide and that was producedaccording to the disclosed methods, wherein the antibody comprises adetectable label; and (c) detecting the detectable label, whereby aSLC26 polypeptide in a sample is detected.

Techniques for detecting such antibody-antigen conjugates or complexesare known in the art and include but are not limited to centrifugation,affinity chromatography and other immunochemical methods. See e.g.,Manson (1992) Immunochemical Protocols. Humana Press, Totowa, N.J.,United States of America; Ishikawa (1999) Ultrasensitive and RapidEnzyme Immunoassay. Elsevier, Amsterdam/N.Y., United States of America;Law (1996) Immunoassay: A Practical Guide. Taylor & Francis,London/Bristol, Pennsylvania, United States of America; Chan (1996)Immunoassay Automation: An Updated Guide to Systems. Academic Press, SanDiego; Liddell & Weeks (1995) Antibody Technology. Bios ScientificPublishers, Oxford, United Kingdom; Masseyeff et al. (1993) Methods ofImmunological Analysis. VCH Verlagsgesellschaft/VCH Publishers,Weinheim, Federal Republic of Germany/N.Y., United States of America;Walker & Rapley (1993) Molecular and Antibody Probes in Diagnosis.Wiley, Chichester, N.Y.; Wyckoff et al. (1985) Diffraction Methods forBiological Macromolecules. Academic Press, Orlando, Fla., United Statesof America; and references cited therein.

In another embodiment of the invention, a modulator that shows specificbinding to a SLC26 polypeptide is used to detect a SLC26 aniontransporter. Analogous to detection of a SLC26 polypeptide using anantibody, the method comprises: (a) obtaining a biological samplecomprising peptidic material; (b) contacting the biological sample witha modulator of a SLC26 polypeptide, wherein the modulator comprises adetectable label; and (c) detecting the detectable label, whereby aSLC26 polypeptide in a sample is detected. Any suitable detectable labelcan be used, for example a fluorophore or epitope label.

IX. Therapeutic Applications

The present invention provides methods for identification of modulatorsof anion transport activity via SLC26A6, SLC26A1, and SLC26A2.Alternatively, a construct encoding a recombinant SLC26 polypeptide ofthe invention can be used to replace diminished or lost SLC26 function.The modulators and constructs of the invention are useful for regulationof anion transport in a subject, for example to remedy dysfunctionalanion transport associated with sulphate homeostasis, sulphation,oxalate homeostasis, transepithelial salt transport, bicarbonatetransport, and physiological pH regulation.

The term “subject” as used herein includes any vertebrate species,preferably warm-blooded vertebrates such as mammals and birds. Moreparticularly, the methods of the present invention are contemplated forthe treatment of tumors in mammals such as humans, as well as thosemammals of importance due to being endangered (such as Siberian tigers),of economical importance (animals raised on farms for consumption byhumans) and/or social importance (animals kept as pets or in zoos) tohumans, for instance, carnivores other than humans (such as cats anddogs), swine (pigs, hogs, and wild boars), ruminants and livestock (suchas cattle, oxen, sheep, giraffes, deer, goats, bison, and camels), andhorses. Also contemplated is the treatment of birds, including thosekinds of birds that are endangered or kept in zoos, as well as fowl, andmore particularly domesticated fowl or poultry, such as turkeys,chickens, ducks, geese, guinea fowl, and the like, as they are also ofeconomical importance to humans.

Functional characterization of SLC26A6, as disclosed herein, indicatesthat it can mediate Cl⁻-formate exchange, Cl⁻—Cl⁻ exchange, SO₄²⁻-exchange, Cl⁻-oxalate exchange, and Cl⁻—HCO₃ ⁻ exchange. The aniontransport properties of SLC26A6 point to its role in a variety ofphysiological functions, including but not limited to regulation ofsulphate homeostasis, oxalate secretion, transepithelial saltabsorption, and bicarbonate transport, including CFTR-dependentbicarbonate transport, as described further herein below.

In one embodiment of the invention, modulators of SLC26A6 activity canbe used for the treatment or prevention of conditions of disrupted aniontransport. In another embodiment of the invention, modulators of SLC26A6expression can be used to treat conditions or pathologies resulting fromlow levels of SLC26A6 expression and/or low levels of SLC26A6 activity.A modulator that enhances SLC26A6 expression can be identified using themethods disclosed herein above. Alternatively, a construct encoding arecombinant human SLC26A6 polypeptide can be used to replace diminishedor lost SLC26A6 function.

The present invention also provides that SLC26A1 does not transport Cl⁻or formate, but does transport SO₄ ²⁻ and oxalate. The anion transportactivities of SLC26A1 are in close agreement with those found in renalproximal tubule basolateral membrane vesicles and are important forsulphate and oxalate homeostasis, as described further herein below.Thus, SLC26A1 modulators, identified by the methods of the presentinvention can also be used for the treatment or prevention of conditionsof disrupted anion transport.

Also disclosed herein is the observation that transport of SO₄ ²⁻ andoxalate by SLC26A1 is strongly activated by impermeant, monovalentanions such as Cl⁻ and formate. Activation by impermeant monovalentanions appears not to be simply a matter of the valence of anioniccharge, since HCO₃ ⁻ does not activate SO₄ ²⁻ uptake by rat SLC26A1(Satoh et al., 1998). This feature of transport activation isdramatically different from that of other SLC26 anion exchangers,including the closely related transporter SLC26A2 (Satoh et al., 1998),and identifies a new approach for modulating SLC26A1 activity.

The present invention further provides novel observations of aniontransport via SLC26A2, a sulphate transporter that contributes to normalsulphation of proteoglycans in bone. Thus, SLC26A2 modulators, includingpH modifiers, can be used to modulate SLC26A2 activity to therebyfacilitate bone health, and to treat or prevent bone disease, asdescribed further herein below.

The present invention still further provides novel methods formodulating anion transport via SLC26A6 and SLC26A2 by regulating pH. Inparticular, acidification of a cellular environment can be used toselectively activate anion transport. For example, modulation ofextracellular pH to about 6.0 can be used to activate Cl⁻—HCO₃ ⁻exchange by SLC26A6. SO₄ ²⁻ transport via SLC26A2 is also stronglyactivated by an acid-outside pH gradient, particularly in the presenceof extracellular Cl⁻.

IX.A. Sulphate Homeostasis

The homeostasis of inorganic sulphate, a physiological anion that isutilized in conjugation reactions of exogenous and endogenous compounds,is maintained by intracellular hydrolysis of sulfoconjugates, oxidationof reduced organic sulfur, and transport of sulphate from extracellularfluids. Sulphate absorption from the gastrointestinal tract andreabsorption by the renal tubules are critical mechanisms formaintenance of sulphate levels.

Sulphate uptake is initiated across the brush-border membrane (BBM) bysodium-dependent sulphate cotransport driven by a lumenal membranesodium gradient. For a review, see Beck & Silve (2001) Kidney Int59:835-845. In addition to the sodium-dependent cotransport system,studies in BBM vesicles have suggested that sulphate can be transportedby an anion exchange mechanism (Karniski & Aronson, 1987; Pritchard,1987; Talor et al., 1987). The transport properties of the BBMsulphate/anion transporter are in close agreement with those of SLC26A6,as disclosed herein.

Exit of sulphate across the basolateral membrane (BLM) occurs via asulphate anion exchanger, which completes the process of transcellularsulphate reabsorption in the proximal tubule. Sulphate transport acrossthe BLM can utilize hydroxyl ions, bicarbonate, and oxalate ascounterions (Low et al., 1984; Pritchard, 1987). These transportcharacteristics are in close agreement with those displayed by SLC26A1,as disclosed herein.

Slight imbalances in sulphate homeostasis can lead to clinicalmanifestations, including hyposulphatemia, hypersulphatemia, and alteredsulphate metabolism. Representative syndromes/disease in which theformation of sulphate ion or the metabolism of oxidized sulfur isdisturbed include Hunter's syndrome, Morquio's syndrome, Maroteaux-Lamysyndrome, metachromatic leuokodystrophy, and multiple-sulfohydrolasedeficiency (Tallgren, 1980).

Increased serum levels of sulphate (hypersulphatemia) are observed inpatients suffering from chronic renal failure. Increased serum sulphatecan alter the sulphation of many endogenous substances and hormones(Falany, 1997; Coughtrie et al., 1998). In most cases, the sulphation ofthese compounds leads to an increase in their urinary excretion (Falany,1997). Excess sulphate can also lead to a reduction in ionized calcium,thereby contributing to the pathogenesis of renal osteodystrophy(Michalk et al., 1990).

Exogenous substances can also disrupt renal handling of sulphate. Forexample, chronic exposure of heavy metals (e.g., mercury, cadmium, lead,and chromium) can lead to nephrotoxicity and hepatotoxicity. Heavymetals can cause cellular necrosis as well as altered absorptiveproperties in the kidney, causing proteinuria, glucosuria,aminoaciduria, calciuria, phophaturia, and sulfaturia (Vacca et al.,1986; Miura et al., 2000). In particular, maximal sulphate transport viaSLC26A1 is strongly inhibited by mercury (Markovich & James, 1999).

The properties of SO₄ ²⁻ transport via SLC26A6, as disclosed herein, arealso similar to those described in placenta (Grassl, 1996), lung(Mohapatra et al., 1993), and pancreas (Elgavish & Meezan, 1992).SLC26A6 is expressed in each of these tissues, suggesting that SLC26A6can mediate the observed transport. SLC26A1 activity has also beendemonstrated in brain, where it is proposed to contribute to myelinsulphation (Lee et al., 1999a).

IX.B. Oxalate Homeostasis

The ability of SLC26A1 and SLC26A6 to mediate oxalate exchange suggestsimportant roles for these transporters in oxalate homeostasis. Apicaloxalate transport by a DIDS-sensitive anion transporter functions inconcert with basolateral oxalate transport mediated by SLC26A1 (Sat-1)to secrete oxalate from the proximal tubule (Senekjian & Weinman, 1982).Of note, sulphate transport by rat DTDST is cis-inhibited by oxalate,consistent with oxalate transport by this SLC26 protein (Satoh, 1998).

Similar data has been reported for SLC26A3 (DRA), although the absolutevalue of heterologous expression was extremely low (Moseley, 1999).SLC26A2 and SLC26A3 are also expressed in the intestine (Haila, 2000;Haila, 2001; Silberg, 1995), where they can play a role in intestinaloxalate transport.

SLC26A6 is expressed at the apical membrane of the proximal tubule(Knauf et al., 2001). The anion transport properties of SLC26A6,disclosed herein, are consistent with the role of SLC26A6 as the apicalrenal oxalate transporter. As disclosed herein, SLC26A6 is stronglyexpressed in small intestine, and thus can also mediate oxalatetransport in gut.

Hyper-excretion of oxalate is an important factor in the pathogenesis ofrenal stones, and increased red cell oxalate transport has been shown tosegregate with oxalate excretion in kindreds with nephrolithiasis(Baggio et al., 1986). Dietary absorption of oxalate is an importantdeterminant of urinary excretion (Holmes et al., 2001).

Based on the foregoing evidence, variation in the human SLC26A6 gene isimplicated as a risk factor for nephrolithiasis. Similarly, modulatorsof SLC26A6 and SLC26A1, identified as disclosed herein, can be used totreat and/or prevent nephrolithiasis.

IX.C. Transepithelial Sodium Absorption

Na⁺—H⁺ exchange mediated by NHE-3 (Na⁺—H⁺ exchanging protein 3)functions in conjunction with apical chloride-formate exchange tomediate transepithelial reabsorption of Na⁺—Cl⁻ by the kidney proximaltubule (Wang et al., 2001) and by segments of the distal nephron (Wanget al., 1992). Apical Cl⁻-base exchange in renal vesicle and wholetubule preparations is also implicated in transepithelial Na⁺—Cl⁻absorption by the proximal tubule (Kurtz et al., 1994). TranscellularNaCl transport in this nephron segment is believed to be mediated by theconcerted action of an apical Na⁺/H⁺ exchanger and a Cl⁻/OH⁻ exchangerto secrete H⁺ and OH⁻ ions that form H₂O in the tubule lumen.

SLC26A6 protein is detected at the apical membrane of epithelial cells(Lohi et al., 2000), including those of the kidney proximal tubule(Knauf et al., 2001). Based on these observations, SLC26A6 was proposedto be the Cl⁻-formate exchanger of the kidney proximal tubule.

The present invention discloses that SLC26A6 mediates Cl⁻-formateexchange and can thereby mediate transepithelial salt reabsorption inthe proximal tubule. The present invention still further provides thatSLC26A6 can mediate transepithelial salt exchange by Cl⁻-base (Cl⁻—OHand/or Cl⁻—HCO₃ ⁻) exchange. Thus, modulators of SLC26A6, identified asdisclosed herein, are useful for the treatment or prevention of Na⁺absorption.

IX.D. Duodenal Ulcer Disease

Protection of duodenal epithelial cells from lumenal acid is mediated byseveral mechanisms including regulation of intracellular pH andsecretion of bicarbonate from the pancreas and Brunner's glands. In theabsence of such protection, duodenal cells and other cells of the uppergastrointestinal tract are believed to reversibly acidify in thepresence of acidic lumenal contents, thereby injuring the epithelium.Lumenal acid also upregulates other putative defense mechanisms, such asmucosal blood flow and mucus gel secretion, suggesting that regulationof bicarbonate levels is part of a multi-component defensive system. SeeAkiba et al. (2001) J Clin Invest 108:1807-1816; Flemstrom & Isenberg(2001) News Physiol Sci 16:23-28; and references cited therein.

The present invention provides that SLC26A6 can mediate Cl⁻-base (Cl⁻—OHand/or Cl⁻—HCO₃ ⁻) exchange. SLC26A6 protein is detected in duodenum(Wang, 2002), consistent with a role for SLC26A6 in Cl⁻—HCO₃ ⁻) exchangein gut. Thus, modulators of SLC26A6 can be used to regulate acid levelsin the gut to thereby treat or prevent conditions such as duodenal ulcerdisease.

IX.E. Cystic Fibrosis

The role of SLC26A6 in Cl⁻—HCO₃ ⁻ exchange is also relevant to thephysiology of tissues that excrete HCO₃ ⁻ under the influence of CFTR, achloride channel whose dysfunction results in cystic fibrosis. Inparticular, Cl⁻-base exchange by SLC26A6 is characteristic of the apicalCFTR-dependent bicarbonate transporter in lung (Lee et al., 1998),submandibular gland (Lee et al., 1999b), and exocrine pancreas (Lee etal., 1999b; Choi et al., 2001). Studies of cystic fibrosis pancreaticcell lines have shown that expression of wild type CFTR can elicit anincrease in SLC26A6 transcripts, a 10-fold activation of DIDS-sensitivesulphate transport, and elevated levels of Cl⁻—HCO₃ ⁻ exchange (Elgavish& Meezan, 1992; Greeley et al., 2001). In addition, the inability ofCFTR mutants to regulate Cl⁻—HCO₃ ⁻ exchange is correlated with thepancreatic insufficiency (Choi et al., 2001).

The present invention provides that SLC26A6 can mediate Cl⁻-base (Cl⁻—OHand/or Cl⁻—HCO₃ ⁻) exchange. Thus, modulators of SLC26A6 can be used toactivate Cl⁻—HCO₃ ⁻ exchange in CF patients.

IX.F. Chondrodysplasia

SLC26A2 (also called DTDS™) encodes an anion transporter whose abnormalfunction can result in any one of several chondrodysplasias, includingdiastrophic dysplasia, astelogenesis type 2, achrondrogenesis type 1B,and multiple ephiphyseal dysplasia (Hastbacka et al., 1994;Superti-Furga et al., 1996; Newbury-Ecob, 1998). Biochemical studies ofpatients with these disorders revealed defects in sulphate uptake, thepresence of undersulphated proteoglycans, and a reduced rate of sulphateincorporation into chondroitin sulphate. In addition, more significantlyreduced levels of proteoglycan sulphation are correlated with clinicalseverity. See Everett & Green (1999) Hum Mol Genet 8:1883-1891 andreferences cited therein. Thus clinical strategies for the treatment ofchondroplasia can be directed toward restoring sulphate transportmediated by SLC26A2 to normal levels. Thus, modulators of SLC26A2, inparticular pH modifiers, can be used to prevent or to treat bonedisorders associated with poor proteoglycan sulphation.

X. Compositions and Therapeutic Methods

In accordance with the methods of the present invention, a compositionthat is administered to alter anion transport activity in a subjectcomprises: (a) an effective amount of a SLC26 modulator; and (b) apharmaceutically acceptable carrier. A SLC26 modulator can comprise anyone of the types of test substances described herein above. A SLC26modulator can also comprise a pH modifier.

The present invention also provides methods for modulating aniontransport activity in a subject via administration of a gene therapyconstruct comprising an SLC26 polypeptide. Such a construct can beprepared as described herein above, further comprising a carriersuitable for administration to a subject.

X.A. pH Modifiers

In one embodiment of the invention, a method is provided for modulatingSLC26 anion transport by altering pH. The disclosure of the presentinvention shows that Cl⁻—HCO₃ ⁻ exchange via SLC26A6 is activated by anacid-outside environment, for example an extracellular pH of about 6.SO₄ ²⁻ transport via SLC26A2 is similarly activated by an acid-outsidepH. Thus, the present invention provides a method for activating aniontransport in a subject, the method comprising administering a modulatorof a SLC26 polypeptide to the subject, wherein the modulator comprises apH modifier.

The term “pH modifier” refers to any substance that can be used toregulate the pH of an in situ environment. An effective amount of a pHmodifier comprises an amount sufficient to alter a pH to a levelsufficient for activation of a SLC26 polypeptide. An effective amount ofa pH modifier effective to achieve the desired in vivo pH modificationwill depend on the acidity or basicity (pKa or pKb) of the compoundused, the pH of the carrier (e.g., a polymer composition) used when invivo, and the in vivo environment's physiologic pH.

Representative pH modifiers include acidic compounds or anhydrousprecursors thereof, or chemically protected acids. For example, a pHmodifier can comprise at least one member selected from the groupconsisting of: amino acids; carboxylic acids and salts thereof; di-acidsand salts thereof; poly-acids and salts thereof; esters that are easilyhydrolyzable in vivo; lactones that are easily hydrolyzable in viva;organic carbonates; enolic compounds; acidic phenols; polyphenoliccompounds; aromatic alcohols; ammonium compounds or salts thereof;boron-containing compounds; sulfonic acids and salts thereof; sulfinicacids and salts thereof; phosphorus-containing compounds; acid halides;chloroformates; acid gases; acid anhydrides; inorganic acids and saltsthereof; and polymers having functional groups of at least one of thepreceding members. A pH modifier of this invention can also comprise atleast one member selected from the group consisting of: glycine;alanine; proline; lysine; glutaric acid; D-galacturonic acid; succinicacid; lactic acid; glycolic acid; poly(acrylic acid); sodium acetate;diglycolic anhydride; succinic anhydride; citraconic anhydride; maleicanhydride; lactide; diethyl oxalate; Meldrum's acid; diethyl carbonate;dipropyl carbonate; diethyl pyrocarbonate; diallyl pyrocarbonate;di-tert-butyl dicarbonate; ascorbic acid; catechin; ammonium chloride;D-glucosamine hydrochloride; 4-hydroxy-ephedrine hydrochloride; boricacid; nitric acid; hydrochloric acid; sulfuric acid; ethanesulfonicacid; and p-toluenesulfonic acid; 2-aminoethylphosphoric acid;methylphosphonic acid; dimethylphosphinic acid; methyl chloroformate;sulfur dioxide; and carbon dioxide.

A pH modifier can be prepared in a micorcapsule, such that the pHmodifier diffuses through the microcapsule or is released by bioerosionof the microcapsule. The microcapsule may be formulated so that the pHmodifier is released from the microcapsule continuously over a period oftime. Microencapsulation of the pH modifier can be achieved by manyknown microencapsulation techniques, as described further herein belowunder the heading “Carriers.”

X.B. Carriers

Any suitable carrier that facilitates preparation and/or administrationof a SLC26 modulator can be used. The carrier can be a viral vector or anon-viral vector. Suitable viral vectors include adenoviruses,adeno-associated viruses (AAVs), retroviruses, pseudotyped retroviruses,herpes viruses, vaccinia viruses, Semiliki forest virus, andbaculoviruses.

Suitable non-viral vectors that can be used to deliver a SLC26polypeptide or a SLC26 modulator include but are not limited to aplasmid, a nanosphere (Manome et al., 1994; Saltzman & Fung, 1997), apeptide (U.S. Pat. Nos. 6,127,339 and 5,574,172), a glycosaminoglycan(U.S. Pat. No. No. 6,106,866), a fatty acid (U.S. Pat. No. 5,994,392), afatty emulsion (U.S. Pat. No. 5,651,991), a lipid or lipid derivative(U.S. Pat. No. 5,786,387), collagen (U.S. Pat. No. 5,922,356), apolysaccharide or derivative thereof (U.S. Pat. No. 5,688,931), ananosuspension (U.S. Pat. No. 5,858,410), a polymeric micelle orconjugate (Goldman et al., 1997) and U.S. Pat. Nos. 4,551,482,5,714,166, 5,510,103, 5,490,840, and 5,855,900), and a polysome (U.S.Pat. No. 5,922,545).

Where appropriate, two or more types of carriers can be used together.For example, a plasmid vector can be used in conjunction with liposomes.

A carrier can be selected to effect sustained bioavailability of a SLC26modulator to a site in need of treatment. The term “sustainedbioavailability” encompasses factors including but not limited toprolonged release of a SLC26 modulator from a carrier, metabolicstability of a SLC26 modulator, systemic transport of a compositioncomprising a SLC26 modulator, and effective dose of a SLC26 modulator.

Representative compositions for sustained bioavailability can includebut are not limited to polymer matrices, including swelling andbiodegradable polymer matrices, (U.S. Pat. Nos. 6,335,035; 6,312,713;6,296,842; 6,287,587; 6,267,981; 6,262,127; and 6,221,958),polymer-coated microparticles (U.S. Pat. Nos. 6,120,787 and 6,090,925) apolyol:oil suspension (U.S. Pat. No. 6,245,740), porous particles (U.S.Pat. No. 6,238,705), latex/wax coated granules (U.S. Pat. No.6,238,704), chitosan microcapsules, and microsphere emulsions (U.S. Pat.No. 6,190,700).

Microcapsules. Microencapsulation can be carried out by dissolving acoating polymer in a volatile solvent, e.g., methylene chloride, to apolymer concentration of about 6% by weight; adding a pH modifyingcompound (selected to be acidic or basic according to the pH level to beachieved in situ) in particulate form to the coating polymer/solventsolution under agitation, to yield a pH modifier concentration of 2% to10% by weight; adding the resulting polymer dispersion to a methylenechloride solution containing a phase inducer, such as silicone oil,under agitation; allowing the mixture to equilibrate for about 20minutes; further adding the mixture slowly to a non-solvent, such asheptane, under rapid agitation; allowing the more volatile solvent toevaporate under agitation; removing the agitator; separating the solidsfrom the silicone oil and heptane; and washing and drying themicrocapsules. The size of the microcapusles will range from about 0.001to about 1000 microns. See e.g., U.S. Pat. No. 6,061,581.

A microencapsulating coating polymer is preferably biodegradable and/orcan permit diffusion of the encapsulated modulator (e.g., a pHmodifier). A microencapsulating coating also preferably has low inherentmoisture content. Biodegradation preferably occurs at rates greater thanor similar to the rate of degradation of the base polymer.

Examples of coating materials that can be used to microencapsulate aSLC26 modulator, for example a pH modifier, include but are not limitedto polyesters, such as polyglycolic acid, polylactic acid, copolymers ofpolyglycolic acid and polylactic acid, polycaprolactone,poly-β-hydroxybutyrate, copolymers of ε-caprolactone andδ-valerolactone, copolymers of ε-caprolactone and DL-dilactide, andpolyester hydrogels; polyvinylpyrrolidone; polyamides; gelatin; albumin;proteins; collagen; poly(orthoesters); poly(anhydrides);poly(alkyl-2-cyanoacrylates); poly(dihydropyrans); poly(acetals);poly(phosphazenes); poly(urethanes); poly(dioxinones); cellulose; andstarches.

Viral Gene Therapy Vectors. Viral vectors of the invention arepreferably disabled, e.g. replication-deficient. That is, they lack oneor more functional genes required for their replication, which preventstheir uncontrolled replication in vivo and avoids undesirable sideeffects of viral infection. Preferably, all of the viral genome isremoved except for the minimum genomic elements required to package theviral genome incorporating the therapeutic gene into the viral coat orcapsid. For example, it is desirable to delete all the viral genomeexcept: (a) the Long Terminal Repeats (LTRs) or Invented TerminalRepeats (ITRs); and (b) a packaging signal. In the case of adenoviruses,deletions are typically made in the E1 region and optionally in one ormore of the E2, E3 and/or E4 regions. Other viral vectors can besimilarly deleted of genes required for replication. Deletion ofsequences can be achieved by a recombinant approach, for example,involving digestion with appropriate restriction enzymes, followed byre-ligation. Replication-competent self-limiting or self-destructingviral vectors can also be used.

Nucleic acid constructs of the invention can be incorporated into viralgenomes by any suitable approach known in the art. Typically, suchincorporation is performed by ligating the construct into an appropriaterestriction site in the genome of the virus. Viral genomes can then bepackaged into viral coats or capsids using any suitable procedure. Inparticular, any suitable packaging cell line can be used to generateviral vectors of the invention. These packaging lines complement thereplication-deficient viral genomes of the invention, as they include,for example by incorporation into their genomes, the genes that havebeen deleted from the replication-deficient genome. Thus, the use ofpackaging lines allows viral vectors of the invention to be generated inculture.

Suitable packaging lines for retroviruses include derivatives of PA317cells, ψ-2 cells, CRE cells, CRIP cells, E-86-GP cells, and 293GP cells.Line 293 cells are preferred for use with adenoviruses andadeno-associated viruses.

Plasmid Gene Therapy Vectors. A SLC26 modulator or SLC26 polypeptide canalso be encoded by a plasmid. Advantages of a plasmid carrier includelow toxicity and easy large-scale production. A polymer-coated plasmidcan be delivered using electroporation as described by Fewell et al.(2001) Mol Ther 3:574-583. Alternatively, a plasmid can be combined withan additional carrier, for example a cationic polyamine, a dendrimer, ora lipid, that facilitates delivery. See e.g., Baher et al. (1999)Anticancer Res 19:2917-2924; Maruyama-Tabata et al. (2000) Gene Ther7:53-60; and Tam et al. (2000) Gene Ther 7:1867-1874.

Liposomes. A composition of the invention can also be delivered using aliposome. Liposomes can be prepared by any of a variety of techniquesthat are known in the art. See e.g., - - - (1997). Current Protocols inHuman Genetics on CD-ROM. John Wiley & Sons, New York; Lasic & Martin(1995) STEALTH® Liposomes. CRC Press, Boca Raton, Fla., United States ofAmerica; Janoff (1999) Liposomes: Rational Design. M. Dekker, New York;Gregoriadis (1993) Liposome Technology, 2nd ed. CRC Press, Boca Raton,Fla., United States of America; Betageri et al. (1993) Liposome DrugDelivery Systems. Technomic Pub., Lancaster; Pa., United States ofAmerica.; and U.S. Pat. Nos. 4,235,871; 4,551,482; 6,197,333; and6,132,766. Temperature-sensitive liposomes can also be used, for exampleTHERMOSOMES™ as disclosed in U.S. Pat. No. 6,200,598. Entrapment of aSLC26 modulator or a SLC26 polypeptide within liposomes of the presentinvention can be carried out using any conventional method in the art.In preparing liposome compositions, stabilizers such as antioxidants andother additives can be used.

Other lipid carriers can also be used in accordance with the claimedinvention, such as lipid microparticles, micelles, lipid suspensions,and lipid emulsions. See e.g., Labat-Moleur et al. (1996) Gene Therapy3:1010-1017; and U.S. Pat. Nos. 5,011,634; 6,056,938; 6,217,886;5,948,767; and 6,210,707.

X.B. Targeting Ligands

As desired, a composition of the invention can include one or moreligands having affinity for a specific cellular marker to therebyenhance delivery of a SLC26 modulator or a SLC26 polypeptide to a sitein need of treatment in a subject. Ligands include antibodies, cellsurface markers, peptides, and the like, which act to home thetherapeutic composition to particular cells.

The terms “targeting” and “homing”, as used herein to describe the invivo activity of a ligand following administration to a subject, eachrefer to the preferential movement and/or accumulation of a ligand in atarget tissue (e.g., a tumor) as compared with a control tissue.

The term “target tissue” as used herein refers to an intended site foraccumulation of a ligand following administration to a subject. Forexample, the methods of the present invention employ a target tissuecomprising a tumor. The term “control tissue” as used herein refers to asite suspected to substantially lack binding and/or accumulation of anadministered ligand.

The terms “selective targeting” of “selective homing” as used hereineach refer to a preferential localization of a ligand that results in anamount of ligand in a target tissue that is about 2-fold greater than anamount of ligand in a control tissue, more preferably an amount that isabout 5-fold or greater, and most preferably an amount that is about10-fold or greater. The terms “selective targeting” and “selectivehoming” also refer to binding or accumulation of a ligand in a targettissue concomitant with an absence of targeting to a control tissue,preferably the absence of targeting to all control tissues.

The terms “targeting ligand” and “targeting molecule” as used hereineach refer to a ligand that displays targeting activity. Preferably, atargeting ligand displays selective targeting. Representative targetingligands include peptides and antibodies.

The term “peptide” encompasses any of a variety of forms of peptidederivatives, that include amides, conjugates with proteins, cyclizedpeptides, polymerized peptides, conservatively substituted variants,analogs, fragments, peptoids, chemically modified peptides, and peptidemimetics. Representative peptide ligands that show tumor-bindingactivity include, for example, those described in U.S. Pat. Nos.6,180,084 and 6,296,832.

The term “antibody” indicates an immunoglobulin protein, or functionalportion thereof, including a polyclonal antibody, a monoclonal antibody,a chimeric antibody, a hybrid antibody, a single chain antibody (e.g., asingle chain antibody represented in a phage library), a mutagenizedantibody, a humanized antibody, and antibody fragments that comprise anantigen binding site (e.g., Fab and Fv antibody fragments). See U.S.Pat. Nos. 5,111,867; 5,632,991; 5,849,877; 5,948,647; 6,054,561 and PCTInternational Publication No. WO 98/10795.

Antibodies, peptides, or other ligands can be coupled to drugs (e.g., aSLC26 modulator or a gene therapy construct comprising a SLC26polypeptide) or drug carriers using methods known in the art, includingbut not limited to carbodiimide conjugation, esterification, sodiumperiodate oxidation followed by reductive alkylation, and glutaraldehydecrosslinking. See e.g., Bauminger & Wilchek (1980) Methods Enzymol70:151-159;

-   -   Goldman et al. (1997) Cancer Res 57:1447-1451; Kirpotin et        al. (1997) Biochemistry 36:66-75; - - - (1997). Current        Protocols in Human Genetics on CD-ROM. John Wiley & Sons, New        York; Neri et al. (1997) Nat Biotechnol 15:1271-1275; Park et        al. (1997) Cancer Lett 118:153-160; and Pasqualini et al. (1997)        Nat Biotechnol 15:542-546; U.S. Pat. No. 6,071,890; and European        Patent No. 0 439 095. Alternatively, pseudotyping of a        retrovirus can be used to target a virus towards a particular        cell (Marin et al., 1997).

X.C. Formulation

Suitable formulations for administration of a composition of theinvention to a subject include aqueous and non-aqueous sterile injectionsolutions which can contain anti-oxidants, buffers, bacteriostats,bactericidal antibiotics and solutes which render the formulationisotonic with the bodily fluids of the intended recipient; and aqueousand non-aqueous sterile suspensions which can include suspending agentsand thickening agents. The formulations can be presented in unit-dose ormulti-dose containers, for example sealed ampoules and vials, and can bestored in a frozen or freeze-dried (lyophilized) condition requiringonly the addition of sterile liquid carrier, for example water forinjections, immediately prior to use. Some preferred ingredients aresodium dodecyl sulphate (SDS), for example in the range of 0.1 to 10mg/ml, preferably about 2.0 mg/ml; and/or mannitol or another sugar, forexample in the range of 10 to 100 mg/ml, preferably about 30 mg/ml;phosphate-buffered saline (PBS), and any other formulation agentsconventional in the art.

The therapeutic regimens and compositions of the invention can be usedwith additional adjuvants or biological response modifiers including,but not limited to, the cytokines interferon alpha (IFN-α), interferongamma (IFN-γ), interleukin 2 (IL2), interleukin 4 (IL4), interleukin 6(IL6), tumor necrosis factor (TNF), or other cytokine affecting immunecells.

X.D. Dose and Administration

A composition of the present invention can be administered to a subjectsystemically, parenterally, or orally. The term “parenteral” as usedherein includes intravenous injection, intramuscular injection,intra-arterial injection, and infusion techniques. For delivery ofcompositions to pulmonary pathways, compositions can be administered asan aerosol or coarse spray. A delivery method is selected based onconsiderations such as the type of the type of carrier or vector,therapeutic efficacy of the composition, and the condition to betreated.

Preferably, an effective amount of a composition of the invention isadministered to a subject. For example, an “effective amount” is anamount of a composition sufficient to modulate SLC26 anion transportactivity.

Actual dosage levels of active ingredients in a therapeutic compositionof the invention can be varied so as to administer an amount of thecomposition that is effective to achieve the desired therapeuticresponse for a particular subject. The selected dosage level will dependupon a variety of factors including the activity of the therapeuticcomposition, formulation, the route of administration, combination withother drugs or treatments, the disease or disorder to be treated, andthe physical condition and prior medical history of the subject beingtreated. Determination and adjustment of an effective amount or dose, aswell as evaluation of when and how to make such adjustments, are knownto those of ordinary skill in the art of medicine.

For local administration of viral vectors, previous clinical studieshave demonstrated that up to 10¹³ pfu (plaque forming units) of viruscan be injected with minimal toxicity. In human patients, 1×10⁹-1×10¹³pfu are routinely used. See Habib et al. (1999) Hum Gene Ther10:2019-2034. To determine an appropriate dose within this range,preliminary treatments can begin with 1×10⁹ pfu, and the dose level canbe escalated in the absence of dose-limiting toxicity. Toxicity can beassessed using criteria set forth by the National Cancer Institute andis reasonably defined as any grade 4 toxicity or any grade 3 toxicitypersisting more than 1 week. Dose is also modified to maximize thedesired modulation of anion transporter activity.

For soluble formulations of a composition of the present invention,conventional methods of extrapolating human dosage are based on dosesadministered to a murine animal model can be carried out using theconversion factor for converting the mouse dosage to human dosage: DoseHuman per kg=Dose Mouse per kg×12 (Freireich et al., 1966). Drug dosesare also given in milligrams per square meter of body surface areabecause this method rather than body weight achieves a good correlationto certain metabolic and excretionary functions. Moreover, body surfacearea can be used as a common denominator for drug dosage in adults andchildren as well as in different animal species as described byFreireich et al. (1966) Cancer Chemother Rep 50:219-244. Briefly, toexpress a mg/kg dose in any given species as the equivalent mg/m² dose,the dose is multiplied by the appropriate km factor. In adult humans,100 mg/kg is equivalent to 100 mg/kg×37 kg/m²=3700 mg/m².

For additional guidance regarding dose, see Berkow et al. (1997) TheMerck Manual of Medical Information, Home ed. Merck ResearchLaboratories, Whitehouse Station, N.J.; Goodman et al. (1996) Goodman &Gilman's the Pharmacological Basis of Therapeutics, 9th ed. McGraw-HillHealth Professions Division, New York; Ebadi (1998) CRC Desk Referenceof Clinical Pharmacology. CRC Press, Boca Raton, Fla., United States ofAmerica; Katzung (2001) Basic & Clinical Pharmacology, 8th ed. LangeMedical Books/McGraw-Hill Medical Pub. Division, New York; Remington etal. (1975) Remington's Pharmaceutical Sciences, 15th ed. Mack Pub. Co.,Easton, Pa.; Speight et al. (1997) Avery's Drug Treatment: A Guide tothe Properties, Choice. Therapeutic Use and Economic Value of Drugs inDisease Management, 4th ed. Adis International, Auckland/Philadelphia,United States of America; Duch et al. (1998) Toxicol Lett100-101:255-263.

EXAMPLES

The following Examples have been included to illustrate modes of theinvention. Certain aspects of the following Examples are described interms of techniques and procedures found or contemplated by the presentco-inventors to work well in the practice of the invention. TheseExamples illustrate standard laboratory practices of the co-inventors.In light of the present disclosure and the general level of skill in theart, those of skill will appreciate that the following Examples areintended to be exemplary only and that numerous changes, modifications,and alterations can be employed without departing from the scope of theinvention.

Example 1 Cloning of Mouse and Human SLC26A6

Human SLC26A6 exons were initially identified in draft sequences of theBAC clone RP11-148G20 and the PAC clone RP4-751E10 by performing tBLASTnsearches of the HTGS database with SLC26A1, SLC26A2, SLC26A3, andSLC26A4 protein sequences as queries.

A BLASTn search of mouse ESTs using the extracted exon contig yielded aSugano mouse I.M.A.G.E. clone (Clone ID No. 2,076,921) having 5′ and 3′EST entries that displayed modest homology to the amino- andcarboxyl-termini of known SLC26 proteins. This full-length mouse SLC26A6cDNA was obtained from Research Genetics, Inc. (Birmingham, Ala., UnitedStates of America) and was sequenced on both strands using fluorescentdye terminator chemistry (available from Applied Biosystems of FosterCity, Calif., United States of America).

A pair of PCR primers (SEQ ID NOs:62-63) was designed using the mousecDNA sequence and human genomic data. The primers were used to clone theopen reading frame of human SLC26A6 from human kidney RNA (availablefrom BD Biosciences Clontech of Palo Alto, Calif., United States ofAmerica).

LA TAQ™ DNA polymerase (TaKaRa of Verivers, Belgium) was used foramplification reactions with the following amplification protocol: 30cycles of denaturation at 98° C. for 30 seconds followed byamplification/extension at 68° C. for 6 minutes. Amplified PCR productswere subcloned in the pCR2.1 vector by the TA CLONING® method(Invitrogen Corporation of Carlsbad, Calif., United States of America).The 3′ UTR was characterized by sequencing a number of human 3′ ESTclones, including I.M.A.G.E. Clone ID Nos: 2,621,351 and 447,726.

A BLASTn search of a mouse genomic database (Celera of Rockville, Md.,United States of America) yielded a 500 kb contig containing the 11 kbmSLC26A6 gene. A subsequent BLASTn search of mouse ESTs was performedusing a 1.7 kb region between the start of EST 2,076,921 and the 3′ UTRof the upstream gene, flamingo 1 (FMI-1)/multiple EGF-like repeatsfactor 2 (MEGF-2). Three 5′ ESTs were identified in the RIKEN database(RIKEN Genome Sciences Center, of Yokohama, Japan), and were determinedto overlap with EST 2,076,921. This alternative 5′ end was cloned byRT-PCR from mouse intestinal total RNA, using a sense primer in exon 1a(SEQ ID NO:64) and an antisense primer in exon 4 (SEQ ID NO:65). Theequivalent human isoforms were cloned by RT-PCR using an exon 1a senseprimer (SEQ ID NO:66) and an exon 3 anti-sense primer (SEQ ID NO:67) andhuman kidney RNA as template.

Analysis of nucleotide and amino acid sequences was performed usingVECTOR NTI® 6.0 software (InforMax, Inc. of Bethesda, Md., United Statesof America), GRAIL® software (Lockheed Martin Energy ResearchCorporation of Oak Ridge, Tenn., United States of America) (Roberts,1991; Xu et al., 1994; Uberbacher et al., 1996) (available athttp://compbio.ornl.gov/Grail-1.3), Phosphobase (Kreegipuu et al., 1999)(http:www.cbs.dtu.dk/databases/PhosphoBase/), TESS (Schug & Overton,1997); available at http://www.cbil.upenn.edu/cgi-bin/tess),Matinspector (Quandt et al., 1995); available from Genomatix SoftwareGmbH of Munich Germany and athttp://transfac.qbf.de/cgi-bin/amtSearch/matsearch.pl), and Prosite(Bucher & Bairoch, 1994; Hofmann et al., 1999)(http://www.expasv.ch/prosite/). A comparison of mouse SLC26A6 and humanSLC26A6 revealed 78% identity at the amino acid level.

The inclusion of exon 1b (FIG. 2) in the longer SLC26A6b transcriptresults in a protein that is 21-23 amino acids shorter than SLC26A6asince this isoform uses a start codon in exon 2. The predicted startcodons in exon 1a and exon 2 include reasonable Kozak sites (Kozak,1996), with purines at position −3 and guanine at position +4. The aminoacid sequence TQALLS (SEQ ID NO:68), which does not contain predictedphosphorylation sites, is conserved in the amino terminal region. MouseSLC26A6b, which lacks the amino terminal extension is functional (FIGS.7-10), and thus the amino terminal sequence is not required fortransport activity.

Example 2 Genomic Localization of SLC26A6

The genomic organization of the chromosomal regions in which mouseSLC26A6 and human SLC26A6 reside is also conserved, such that both genesare flanked on the 5′ ends by the FMI-1/MEGF-2 gene and at the 3′ end bythe UQCRC1 and ColA7 genes (Hoffman et al., 1993; Li et al., 1993).

Human STSs and previously localized genes were used to localize humanSLC26A6 on chromosome 3p21 between markers D3S3582 and D3S1588. Themouse ColA7 gene is positioned about 40 kb 3′ to SLC26A6 within a 500 kbgenomic contig, and thus is physically linked to SLC26A6. ColA7 islocalized on mouse chromosome 9 at 61.0 cm (Li et al., 1993), and thusSLC26A6 is localized at ˜61 cM, which is a syntenic segment of humanchromosome 3p21.

Mouse SLC26A6 and human SLC26A6 share a similar organization,encompassing 21 coding exons and ˜10 kb of genomic DNA. Intron-exonboundaries for mouse SLC26A6 are presented in Table 2 and are set forthas SEQ ID NOs:14-55. Both genes include an alternative 5′ non-codingexon (exon 1b). Non-quantitative RT-PCR (FIG. 5D) suggests that theisoform in which exon 1 b has been spliced out, denoted SLC26A6a, isexpressed at a lower level than SLC26A6b.

Example 3 Northern Analysis of SLC26A6

RNA was extracted from C57BU6J mice and human cell lines using guanidineisothiocyanate and cesium chloride. The human pancreatic Panc-1 cellline and the human pulmonary Calu-3 cell line were obtained from theAmerican Type Culture Collection (ATCC of Manassas, Va., United Statesof America). Calu-3 is a model for pulmonary submucosal gland serousepithelial cells (Lee et al., 1998), and Panc-1 is a model forpancreatic ductal epithelial cells (Elgavish & Meezan, 1992).

Total RNA (10 μg/lane) was size fractionated by electrophoresis (5%formaldehyde, 1% agarose), and transferred to a nylon membrane(Stratagene of La Jolla, Calif., United States of America). The blot washybridized sequentially with ³²P-labeled randomly-primed probescorresponding to full-length GAPDH (glyceraldehyde-3-phosphatedehydrogenase) and a 3′ probe from mSLC26A6 (nucleotides 2239-2673 ofmouse SLC26A6b, SEQ ID NO:7).

Northern blots prepared using 2 μg/lane of poly-A+ RNA were obtainedfrom BD Biosciences Clontech of Palo Alto, Calif., United States ofAmerica. The blots were hybridized to a human SLC26A6a probe(nucleotides 2090-2587 of human SLC26A6a, SEQ ID NO:1) and a β-actinprobe.

Hybridization of all blots was performed overnight at 42° C. inExpress-Hyb solution (Clontech of Palo Alto, Calif., United States ofAmerica). Membranes were washed twice for 10 minutes at room temperaturein 2×SSCP/0.1% SDS, and twice for 1 hour at 65° C. in 0.1×SSCP/0.1% SDS.

Northern blot analysis of human tissues indicated that human SLC26A6 iswidely expressed. A 3.0 kb transcript was detected in kidney, muscle,pancreas, intestine, and heart (FIG. 5A). Minor bands of highermolecular weight (˜4 kb and ˜6 kb) were also detected in several tissuesand are predicted to represent incompletely spliced transcripts. Theseextra bands are unlikely to result from cross-hybridization to otherfamily members, since a non-coding probed was used.

Human SLC26A6 is also robustly expressed in the human Calu-3 and Panc-1cell lines (FIG. 5B). Mouse SLC26A6 is also broadly expressed (FIG. 5C).

The widespread expression of SLC26A6 is consistent with the presence ofa CpG island overlapping exon 1a (FIG. 4), which is conserved in bothhuman SLC26A6 and mouse SLC26A6. The most 5′ mouse SLC26A6a ESTs begin˜100 bp 5′ of the start codon in exon 1a, and thus the transcriptionalstart site lies at or 5′ to the most 5′ site of exon 1a. The genomic DNAflanking mouse SLC26A6 exon 1a suggests that SLC26A6 uses a TATA-lesspromoter that is rich in Sp1 binding sites (FIG. 4).

Example 4 Cloning of SLC26A1

A full-length mouse SLC26A1 cDNA was identified on an EST cDNA(I.M.A.G.E. Clone ID No. 1,450,460). Mouse SLC26A1 protein (FIG. 3) is91% identical to rat SLC26A1 protein, and 76% identical to human SLC26A1protein. Large genomic contigs containing the mouse and human SLC26A 1genes reveal a conserved organization, such that they are both flankedat the 5′ end by the FGFRL-1 (Wiedemann & Trueb, 2001), GAK (Kimura etal., 1997), and DAGK4 (Endele et al., 1996) genes and at the 3′ end bythe L-iduronidase gene. Mouse FGFRL-1 and L-iduronidase have both beenlocalized on mouse chromosome 5 at ˜57 cM (Wiedemann & Trueb, 2001),syntenic with the region of human chromosome 4p16 containing SLC26A1,GAK (Kimura et al., 1997), and DAGK4 (Endele et al., 1996).

The genomic organization of the human and mouse SLC26A1 genes is alsoconserved, although analysis of a number of 5′ mouse SLC26A1 ESTsreveals the existence of two 5′ non-coding exons in the mouse gene. The5′ non-coding exon positioned more 3′ relative to the alternate 5′ exonwas denoted exon 1b. Exon 1b is excluded from a number of ESTsindicating that it is alternatively spliced. The relative position ofthe junction between the two coding exons of SLC26A 1 and SLC26A2(DTST), which together form a separate branch of the gene family, isconserved in the respective mouse and human genes.

Example 5 Expression of SLC26A6 and SLC26A 1 in Xenopus laevis Oocytes

Full-length mouse SLC26A6b and SLC26A 1 cDNAs were cloned into theXenopus expression vector pGEMHE (Liman et al., 1992). The SLC26A6 andSLC26A1 expression constructs were linearized, and cRNA was transcribedin vitro using T7 RNA polymerase and a MMESSAGE MMACHINE® transcriptionkit (Ambion, Inc. of Austin, Tex., United States of America).Defolliculated oocytes were injected with 25 nl to 50 nl of water orwith a solution containing cRNA at a concentration of 0.5 μg/μl (12.5 ngto 25 ng per oocyte) using a Nanoliter-2000 injector (WPI Instruments ofSarasota, Fla., United States of America). Oocytes were incubated at 17°C. in 50% Leibovitz's L-15 media supplemented withpenicillin/streptomycin (1000 units/ml) and glutamine for 2-3 days foruptake assays.

Example 6 Anion Transport

For sulphate uptake assays, oocytes were pre-incubated for 20 minutes inchloride-free uptake medium (100 mM NMDG gluconate, 2 mM potassiumgluconate, 1 mM calcium gluconate, 1 mM magnesium gluconate, 10 mMHEPES-Tris, pH 6.0 or pH 7.5 as indicated), followed by a 60-minuteperiod for uptake in the same medium supplemented with 1 mM K₂ ³⁵SO₄ (40μCi/ml). The cells were then washed three times in uptake buffer with 5mM cold K₂SO₄ to remove tracer activity in the extracellular fluid. Theoocytes were dissolved individually in 10% SDS, and tracer activity wasdetermined by scintillation counting. Uptake of chloride, formate, andoxalate was assayed using the same chloride-free uptake solutions,substituting 8.3 mM ³⁶Cl, 500 μM [¹⁴C]oxalate, or 50 μM [¹⁴C]formate forlabeled sulphate.

For sulphate exchange and cis-inhibition experiments, the concentrationof NMDG-gluconate in the uptake solution was adjusted to maintainisotonic osmolality, which was confirmed experimentally using a FISKE®osmometer (Fiske Associates, Inc. of Bethel, Conn., United States ofAmerica).

SLC26A6 Anion Transport

As shown in FIG. 6A, SLC26A6b transported sulphate independent of Cl⁻(602±0 pmol/oocyte/hour at pH 7.4 versus 2.0±0.2 pmol/oocyte/hour inwater-injected controls). SLC26A6b also transported sulphate independentof Na⁺ (652±57 pmol/oocyte/hour versus 5.8±0.9 pmol/oocyte/hour inwater-injected controls) (FIG. 6A). Sulphate uptake was notsignificantly altered at pH 7.4 versus pH 6.0, although SLC26A6b wasmore sensitive to DIDS at pH 6.0 (101±10 pmol/oocyte/hour) than at pH7.4 (301±24 pmol/oocyte/hour).

SLC26A6b-injected oocytes also retained ³⁶Cl⁻ (FIG. 6B). In this case, aconsistent difference between uptake at pH 7.4 (4345±243pmol/oocyte/hour versus 10±12 pmol/oocyte/hour in water-injectedcontrols) and uptake at pH 6.0 (4193±109 pmol/oocyte/hour versus 99±16pmol/oocyte/hour in water-injected controls) was not observed.

Since the concentrations of SO₄ ²⁻ and Cl⁻ in Xenopus oocytes are about1 mM (Chernova et al., 1997) and about 30 mM (Romero et al., 2000),respectively, a significant component of the measured uptakesrepresented SO₄ ²⁻—SO₄ ²⁻ and Cl⁻—Cl⁻ exchange at the concentrationsused in the extracellular uptake medium (1 mM for SO₄ ²⁻ and 8 mM forCl⁻). SLC26A6b also mediated Cl⁻—HCO₃ ⁻ exchange (FIG. 11), and thus,the observed lack of stimulation by more acidic extracellular medium(FIG. 6B) was surprising.

A shared property of the SLC26A1, SLC26A2, SLC26A3, and SLC26A4exchangers is cis-inhibition by transported substrates (Satoh et al.,1998; Moseley et al., 1999; Scott & Karniski, 2000; Knauf et al., 2001).To assess the repertoire of substrates exchanged with Cl⁻, SLC26A6binjected oocytes were incubated in the presence of sulphate, formate,halides, nitrate, and lactate. Among this group, sulphate, formate,halides, and nitrate, but not lactate significantly inhibited Cl⁻—Cl⁻exchange (FIG. 7A). A similar profile was obtained for SO₄ ²⁻ transport(FIG. 7B).

The observed profile of cis-inhibition was similar to that of the renalCl⁻-formate exchanger (Karniski & Aronson, 1987). As predicted,SLC26A6b-injected oocytes transported both oxalate (487±50pmol/oocyte/hour vs. 12±1 pmol/oocyte/hour in water-injected controls,FIG. 8A) and formate (45±5 pmol/oocyte/hour vs. 7 pmol/oocyte/hour inwater-injected controls, FIG. 8B).

Sulphate exchange was also measured in SLC26A6b-injected oocytes in thepresence of extracellular substrates (Scott & Karniski, 2000), andcis-inhibition was measured in SLC26A6b-injected oocytes in the presenceof sulphate. Since SLC26A4 is known to transport formate and Cl⁻ butneither oxalate (Scott & Karniski, 2000) nor SO₄ ²⁻ (Scott et al.,1999), it was proposed that SLC26A6b does not catalyze formate-SO₄ ²⁻exchange. Surprisingly, SLC26A6b clearly mediated exchange of SO₄ ²⁻with SO₄ ²⁻, Cl⁻, formate and oxalate (FIG. 7C).

SLC26A1 Anion Transport

SLC26A1 mediated SO₄ ²⁻ and oxalate uptake (72±2 pmol/oocyte/h, FIGS. 6Aand 8A), but not formate (7 pmol/oocyte/h). The absolute transport rateswere significantly lower for both SO₄ ²⁻ and oxalate when compared toSLC26A6b-facilitated transport. However, SO₄ ²⁻ transport rates forSLC26A1 were much higher in the presence of extracellular Cl⁻, closer tothe values of SLC26A6 in the absence of Cl⁻.

SLC26A1 mediated minimal Cl⁻—Cl⁻ exchange in either the presence orabsence of 25 mM SO₄ ²⁻ (FIG. 6D), suggesting that Cl⁻ and SO₄ ²⁻ arenot synergistically co-transported by SLC26A1. The lack of Cl⁻—Cl⁻exchange is consistent with transport studies using basolateral membranevesicles from renal cortex, which indicate that the basolateral SO₄²⁻—HCO₃ ⁻ exchanger does not transport Cl⁻ (Kuo & Aronson, 1988).

Several monovalent ions (halides, formate, and lactate) were observed toactivate SO₄ ²⁻ transport via SLC26A1, although Cl⁻ (FIG. 6B) andformate (FIG. 8B) were not transported. In contrast, the divalentsubstrates SO₄ ²⁻ and oxalate were strongly cis-inhibitory to SO₄ ²⁻transport via SLC26A1 (FIG. 9A).

Both I⁻ and Br⁻ were observed to be cis-inhibitory to SO₄ ²⁻ transportand to Cl⁻ transport by SLC26A6, indicating that they are potentialsubstrates of this transporter. The activation of SLC26A1 was not uniqueto SO₄ ²⁻ transport, since oxalate transport in SLC26A 1-injectedoocytes is also higher in the presence of monovalent anions (FIG. 9B).In this experiment, SLC26A6 served as a control because oxalatetransport by oocytes injected with this cRNA was strongly cis-inhibitedby these anions (FIG. 9B).

SLC26A2 Anion Transport

Oocytes expressing mouse SLC26A2 mediated robust ³⁵SO₄ ²⁻ uptake whichis increased significantly at pH 6.0 (FIG. 10A). The addition ofextracellular Cl⁻ significantly inhibited ³⁵SO₄ ²⁻ uptake by SLC26A2(FIG. 10B). SLC26A2-injected oocytes also mediated significant ³⁶Cl⁻,uptake (FIG. 10C), an observation that has not been previously made forthis anion exchanger. Since the concentration of Cl⁻ in Xenopus oocytecytoplasm is ˜30 mM (Romero, 2000), versus 8 mM in the extracellularuptake medium, a significant component of the uptake activity probablyrepresents Cl⁻—Cl⁻ exchange.

Example 7 Electrogenic Transport

For electrophysiological measurements, oocytes were studied 3 days to 11days following injection of SLC26 expression constructs. CO₂/HCO₃-freeND96 medium contained 96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1.8 mM CaCl₂,and 5 mM HEPES (pH 7.5 and 195-200 mOsm). For CO₂/HCO₃-equilibratedsolutions, 33 mM NaHCO₃ replaced 33 mM NaCl. In “O—Na⁺” solutions,choline replaced Na⁺. In “O—Cl⁻” solutions, gluconate replaced Cl. Allsolutions were titrated to pH 7.5, and were continuously bubbled withCO₂-balanced O₂ to maintain pCO₂ and pH. Ion selective microelectrodeswere prepared, calibrated, and employed as described by Romero et al.(1998) Am J Physiol 274:F425-432 and by Romero et al. (2000) J Biol Chem275:24552-24559. All pH electrodes had slopes of at least −56 mwdecadechange.

To determine whether SLC26A6 functions as a Cl⁻—HCO₃ ⁻ exchanger,intracellular pH (pH_(i)) was measured in response to the manipulationof bath HCO₃ ⁻ and Cl⁻. The initial addition of CO₂/HCO₃ ⁻ to the bathsolution resulted in the acidification of oocytes due to CO₂ plasmamembrane diffusion, then intracellular hydration and dissociationforming intracellular H⁺ and HCO₃ ⁻ ions.

FIG. 11A shows that a water-injected oocyte exposed to 5% CO₂/33 mM HCO₃⁻ (pH 7.5) acidified by 0.44 pH units (−0.46±0.01, n=8) at an initialrate of 46×10⁻⁴ pH units/sec (460×10⁻⁵ pH units/sec; −382±19×10⁻⁵ pHunits/sec, n=8). The initial intracellular pH (pH_(i)) ofSLC26A6-injected oocytes was essentially the same as that of watercontrols (water, 7.26±0.03, n=8; SLC26A6, 7.29±0.03, n=10). Addition of5% CO₂/33 mM HCO₃ ⁻ produced a fall in pH_(i) of 0.50 pH units(−0.46±0.02, n=10) at an initial rate of 35×10⁻⁴ pH units/sec (350×10⁻⁵pH units/sec; −387±15×10⁻⁵ pH units/sec, n=10). SLC26A6-injected oocyteswere depolarized (−26.3±4.5 mV, n=10) compared to control oocytes(−44.8±4.3 mV, n=8). The addition of HCO₃ ⁻ produced a slight but abruptdepolarization in SLC26A6-injected oocytes (3.1±0.6 mV, n=9). Cl⁻replacement (gluconate) did not affect pH_(i) of the water control(+6.0±2.2×10⁻⁵ pH units/sec, n=8; FIG. 11A). However, Cl⁻ removalincreased pH_(i) in SLC26A6-injected oocytes at the rate of 44×10⁻⁵ pHunits/sec (+72±8.8×10⁻⁵ pH units/sec, n=10; FIG. 11B), which ceasedafter Cl⁻ re-addition. A plot of pH_(i) and and a plot of V_(m) for anindividual SLC26A6-injected oocyte are shown in FIG. 11B. Surprisingly,gluconate replacement evoked a 37 mV hyperpolarization (−22.7±2.9 mV,n=9; vs.+0.2±2.0 mV, n=8 for controls). A second Cl⁻ removal increasedthe alkalinization rate to 28×10⁻⁵ pH units/sec (+41±6.2×10⁻⁵ pHunits/sec, n=8; FIG. 11B) and reproduced the hyperpolarization(−18.6±3.8 mV, n=8).

FIGS. 11A and 11B illustrate an experiment with individualwater-injected and SLC26A6-injected oocytes. These observations havebeen repeated using SLC26A6-injected oocytes from five separate frogs.In all the experiments using SLC26A6-injected oocytes, the secondalkalinization induced by Cl⁻ removal, which occurs at a higher pHI, hasa lower rate (+72×10⁻⁵ pH units/sec for the first alkalinization, and+41×10⁻⁵ pH units/sec, versus +6.0×10⁻⁵ pH units/sec for the single Cl⁻removal in water-injected oocytes).

In another set of experiments, Na⁺ was replaced with choline to testcation dependence of SLC26A6. Na⁺ removal and replacement did notobviously affect pH_(i). Prior to CO₂ removal, pH_(i) rose to 7.2, whichis approximately the non-HCO₃ ⁻ level. Removal of 5% CO₂/33 mM HCO₃ ⁻elicited a robust alkalinization and pH_(i) overshoot to 7.9 (7.83±0.07,n=10), indicative of cellular HCO₃ ⁻ loading (ΔpH_(i) forSLC26A6-injected oocytes was +0.53±0.07, n=10). This overshoot was notobserved in control oocytes (ΔpH_(i) for controls was +0.02±0.04, n=8).

Since mouse SLC26A6 clearly functions as a Cl⁻—HCO₃ ⁻ exchanger, Cl⁻—OH⁻exchange via SLC26A6 was also tested. For these experiments, non-HCO₃ ⁻solutions were continuously bubbled with 100% O₂. FIGS. 12A and 12Billustrate the non-HCO₃ ⁻ responses of control and SLC26A6-injectedoocytes. Removal of bath Cl⁻ from control oocytes (FIG. 12A) did notchange pH_(i) (−2.1±1.8×10⁻⁵ pH units/sec, n=7) or V_(m) (−0.6±3.5 mV,n=7). In contrast, removal of bath Cl⁻ alkalinized (+27±6.4×10⁻⁵ pHunits/sec, n=6) and hyperpolarized (−7.3±2.8 mV, n=8) SLC26A6-injectedoocytes (FIG. 12B). Cl⁻ re-addition to the bath stopped thealkalinization and returned V_(m) to the initial value.

Example 8 Identification of SLC26 Orthologs from Other Species

A feature of the mammalian SLC26 gene family is relatively lowconservation between orthologs in mouse and man; the percent amino acididentity ranges from a low of 76% (SLC26A8) to a high of 90% (SLC26A9),versus the reported median of 86% for mouse and human orthologous genes.Makalowski, W. & Boguski, 1998. This sequence divergence is reflected infunctional variation, which can be exploited for structure-functionanalysis. Human SLC26A6 and murine Slc26a6 appear to differ in theirfunctional characteristics. Although SLC26A6 clearly transports Cl⁻(FIG. 13), absolute rates of SO₄ ²⁻ transported by this exchanger aremuch lower (˜200 pmol/oocyte/hr vs. ˜4,000 pmol/oocyte/hr) than thosemeasured for Slc26a6 (FIGS. 14 and 15, showing SO₄ ²⁻ uptakes atincreasing concentrations of extracellular SO₄ ²⁻ with stable amount ofradioactive ³⁵SO₄ ²⁻).

While orthologous pairs of murine and human SLC26 exchangers are of usefor structure-function analysis, full-length cDNAs from several otherspecies were also identified. To clone the pig SLC26A6 ortholog,overlapping pig SLC26A6 ESTs in Genbank were used to construct theentire cDNA in silico; this cDNA was then cloned by long-range RT-PCRfrom the LLC-PK1 cell line, a porcine cell-culture model of the renalproximal tubule. The pig SLC26A6a protein is 77% identical to murineSlc26a6 and 80% identical to human SLC26A6. SEQ ID NOs:90-91 are thenucleic acid and amino acid sequences, respectively, of SLC26A6aisolated from pig (Sus scrofa).

Xenopus laevis EST cDNAs derived from the orthologs of several SLC26genes were also identified. This effort has resulted in theidentification of the Xenopus SLC26a6 ortholog (“xSLC26A6”), SLC26A1ortholog (“xSLC26A1”), and three apparent orthologs of SLC26A4 (pendrinor PDS), provisionally denoted “xPDS1-3”. A phylogenetic tree showsthese relationships well (FIG. 16). SEQ ID NOs: 80-85 are the nucleicacid (even SEQ ID NOs:80, 82, and 84) and amino acid sequences (odd SEQID NOs: 81, 83, and 85) of SLC26A4 (PDS1-3). SEQ ID NOs: 86-87 are thenucleic acid and amino acid sequences, respectively, of SLC26A1 isolatedfrom Xenopus laevis. SEQ ID NOs: 88-89 are the nucleic acid and aminoacid sequences, respectively, of SLC26A6 isolated from Xenopus laevis.

One or more of these xPDS genes likely corresponds to the Xenopusdescendant of mammalian SLC26A3. However, the xPDS proteins are muchmore homologous to mammalian SLC26A4/pendrin than to SLC26A3; xPDS1,xPDS2, and xPDS3 are respectively 67%, 59%, and 54% identical to mouseSlc26a4, versus 45%, 42%, and 40% identity to mouse Slc26a3. ThexSLC26A6 exchanger and the three xPDS exchangers are capable of robustCl⁻ transport when expressed in Xenopus oocytes (see FIG. 17). The xPDS2protein can also clearly mediate Cl⁻—HCO₃ ⁻ exchange (see FIG. 18). Asreported for human PDS/SLC26A6 (Scott, D. A., et al., 1999), none of thexPDS clones transport SO₄ ²⁻; these cDNAs will therefore be useful toolsto identify the molecular determinants of monovalent specificity (i.e.domains that impart specificity for monovalent ions) in mammalianSLC26A4 and SLC26A3.

The SLC26A6 exchanger is a likely mediator of intestinal oxalateabsorption and thus a potential therapeutic target in calcium-oxalatestone disease. Intestinal expression of the SLC26A3 or DRA protein isalso robust, in keeping with the genetic role of SLC26A3 in congenitalchloride-losing diarrhea. Hoglund, P. et al., 1996. SLC26A3 has reportedto possess mediate oxalate transport; however, the data is notconvincing (Moseley, R. H. et al., 1999; Silberg, D. G., et al., 1995),and it is difficult to understand why the SLC26A3 protein wouldtransport oxalate when its close homolog SLC26A4 does not. Scott, D. A.,et al., 1999; Scott, D.A. & Karniski, L. P., 2000. Cis-inhibition of therobust Cl transport mediated by SLC26A3-injected oocytes suggests thatthis protein transports minimal if any oxalate (FIG. 19). This suggestsfurthermore that SLC26A6 is the more important apical pathway forintestinal oxalate absorption.

Example 9 Generation of Ortholog-Specific SLC26 Antibodies

To facilitate the understanding of the physiological role of theSLC26A6/Slc26a6 anion exchanger a number of polyclonal antibodies weregenerated against unique epitopes within the murine Slc26a6 sequence.These include both an N-terminal antibody, directed against the sequenceQEQLEDLGHWGPAAKTH (residues 40-56 of the Slc26a6a protein —SEQ ID NO:92)and a C-terminal antibody directed against the sequenceKVHQGEELQDVVSSNQEDA (residues 631-649—SEQ ID NO:93). Both the N- andC-terminal antibodies recognize human SLC26A6 and murine Slc26a6proteins, specifically a “core” (likely high-mannose) protein of −83 kDaand a complex glycoprotein of ˜110 kDa (FIGS. 20 and 21). High-titreantisera have also been obtained for Slc26a1/SLC26A1-specific andSlc26a2/SLC26A2-specific antigens, YRLTGLDAGHSATRKDQ (residues 564-580of Slc26a1—SEQ ID NO:94) and KEQHNVSPRDSAEGNDS (residues 6-22 ofSLC26A2—SEQ ID NO:95).

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It will be understood that various details of the invention can bechanged without departing from the scope of the invention. Furthermore,the foregoing description is for the purpose of illustration only, andnot for the purpose of limitation—the invention being defined by theclaims appended hereto.

1. An isolated polypeptide comprising a functional SLC26A6 polypeptide.2. The functional SLC26A6 polypeptide of claim 1 comprising: (a) apolypeptide encoded by a nucleic acid of any one of odd-numbered SEQ IDNOs:1-7; (b) a polypeptide encoded by a nucleic acid substantiallyidentical to any one of odd-numbered SEQ ID NOs:1-7; (c) a polypeptidecomprising an amino acid sequence of any one of even-numbered SEQ IDNOs:2-8; or (d) a polypeptide substantially identical to any one ofeven-numbered SEQ ID NOs:2-8.
 3. The functional SLC26A6 polypeptide ofclaim 1, wherein the SLC26A6 polypeptide is encoded by an isolatednucleic acid segment selected from the group consisting of: (a) anisolated nucleic acid molecule encoding a polypeptide of any one ofeven-numbered SEQ ID NOs:2-8; (b) an isolated nucleic acid molecule ofany one of odd-numbered SEQ ID NOs:1-7; (c) an isolated nucleic acidmolecule which hybridizes to a nucleic acid sequence of any one ofodd-numbered SEQ ID NOs:1-7 under wash stringency conditions representedby a wash solution having less than about 200 mM salt concentration anda wash temperature of greater than about 45° C., and which encodes afunctional SLC26A6 polypeptide; and (d) an isolated nucleic acidmolecule differing by at least one functionally equivalent codon fromthe isolated nucleic acid molecule of one of (a), (b), and (c) above innucleic acid sequence due to the degeneracy of the genetic code, andwhich encodes a functional SLC26A6 polypeptide encoded by the isolatednucleic acid of one of (a), (b), and (c) above.
 4. The functionalSLC26A6 polypeptide of claim 1, wherein the functional propertycomprises Cl⁻-fomate exchange.
 5. The functional SLC26A6 polypeptide ofclaim 1, wherein the functional property comprises Cl⁻—Cl⁻ exchange. 6.The functional SLC26A6 polypeptide of claim 1, wherein the functionalproperty comprises SO₄ ²⁻ exchange.
 7. The functional SLC26A6polypeptide of claim 1, wherein the functional property comprisesCl⁻-oxalate exchange.
 8. The functional SLC26A6 polypeptide of claim 8,wherein the functional property comprises Cl⁻-base exchange.
 9. Thefunctional SLC26A6 polypeptide of claim 8, wherein the base comprisesHCO₃ ⁻.
 10. The functional SLC26A6 polypeptide of claim 1, wherein theCl⁻-base exchange comprises electrogenic transport.
 11. A system forrecombinant expression of a functional SLC26A6 polypeptide, the systemcomprising: (a) a functional SLC26A6 polypeptide; and (b) a host cellcomprising the functional SLC26A6 polypeptide.
 12. The system of claim11, wherein the host cell comprises a mammalian cell.
 13. The system ofclaim 12, wherein the mammalian cell comprises a human cell.
 14. Anisolated nucleic acid encoding a human SLC26A6a polypeptide.
 15. Theisolated nucleic acid of claim 14, further comprising a nucleic acidencoding a polypeptide of SEQ ID NO:2.
 16. The isolated nucleic acid ofclaim 14, further comprising a nucleic acid of SEQ ID NO:1.
 17. Anisolated human SLC26A6a polypeptide.
 18. The isolated human SLC26A6apolypeptide of claim 17, further comprising: (a) a polypeptide of SEQ IDNO:2; or (b) a polypeptide encoded by a nucleic acid molecule of SEQ IDNO:1.
 19. A system for recombinant expression of a human SLC26A6apolypeptide, the system comprising: (a) a human SLC26A6a polypeptide;and (b) a host cell comprising the human SLC26A6a polypeptide.
 20. Anisolated nucleic acid encoding a mouse SLC26A6 polypeptide.
 21. Theisolated nucleic acid of claim 20, further comprising a nucleic acidencoding a polypeptide of SEQ ID NO:6 or
 8. 22. The isolated nucleicacid of claim 20, further comprising a nucleic acid of SEQ ID NO:5 or 7.23. An isolated mouse SLC26A6 polypeptide.
 24. The isolated mouseSLC26A6 polypeptide of claim 23, further comprising: (a) a polypeptideof SEQ ID NO:6 or 8; or (b) a polypeptide encoded by a nucleic acidmolecule of SEQ ID NO:5 or
 7. 25. A system for recombinant expression ofa mouse SLC26A6 polypeptide, the system comprising: (a) a mouse SLC26A6polypeptide; and (b) a host cell comprising the mouse SLC26A6polypeptide.
 26. A method for identifying a modulator of a SLC26A6polypeptide, the method comprising: (a) providing a recombinantexpression system whereby a functional SLC26A6 polypeptide is expressedin a host cell; (b) providing a test substance to the system of (a); (c)assaying a level or quality of SLC26A6 function in the presence of thetest substance; (d) comparing the level or quality of SLC26A6 functionin the presence of the test substance with a control level or quality ofSLC26A6 function; and (e) identifying a test substance as an aniontransport modulator by determining a level or quality of SLC26A6function in the presence of the test substance as significantly changedwhen compared to a control level or quality of SLC26A6 function.
 27. Themethod of claim 26, wherein the functional SLC26A6 polypeptidecomprises: (a) a polypeptide encoded by a nucleic acid of any one ofodd-numbered SEQ ID NOs:1-7; (b) a polypeptide encoded by a nucleic acidsubstantially identical to any one of odd-numbered SEQ ID NOs:1-7; (c) apolypeptide comprising an amino acid sequence of any one ofeven-numbered SEQ ID NOs:2-8; or (d) a polypeptide substantiallyidentical to any one of even-numbered SEQ ID NOs:2-8.
 28. The method ofclaim 26, wherein the functional SLC26A6 polypeptide is encoded by anisolated nucleic acid segment selected from the group consisting of: (a)an isolated nucleic acid molecule encoding a polypeptide of any one ofeven-numbered SEQ ID NOs:2-8; (b) an isolated nucleic acid molecule ofany one of odd-numbered SEQ ID NOs:1-7; (c) an isolated nucleic acidmolecule which hybridizes to a nucleic acid sequence of any oneodd-numbered SEQ ID NOs:1-7 under wash stringency conditions representedby a wash solution having less than about 200 mM salt concentration anda wash temperature of greater than about 45° C., and which encodes afunctional SLC26A6 polypeptide; and (c) an isolated nucleic acidmolecule differing by at least one functionally equivalent codon fromthe isolated nucleic acid molecule of one of (a), (b), and (c) above innucleic acid sequence due to the degeneracy of the genetic code, andwhich encodes a functional SLC26A6 polypeptide encoded by the isolatednucleic acid of one of (a), (b), and (c) above.
 29. The method of claim26, wherein the host cell comprises a mammalian cell.
 30. The method ofclaim 29, wherein the mammalian cell comprises a human cell.
 31. Themethod of claim 26, wherein the SLC26A6 function comprises Cl⁻-fomateexchange.
 32. The method of claim 26, wherein the SLC26A6 functioncomprises Cl⁻—Cl⁻ exchange.
 33. The method of claim 26, wherein theSLC26A6 function comprises SO₄ ²⁻ exchange.
 34. The method of claim 26,wherein the SLC26A6 function comprises Cl⁻-oxalate exchange.
 35. Themethod of claim 26, wherein the SLC26A6 function comprises Cl⁻-baseexchange.
 36. The method of claim 35, wherein the base comprises HCO₃ ⁻.37. The method of claim 35, wherein the Cl⁻-base exchange compriseselectrogenic transport.
 38. An anion transporter modulator identified bythe method of claim
 26. 39. A method for modulating anion transportactivity in a subject, the method comprising: (a) preparing acomposition comprising a modulator identified according to the method ofclaim 26, and a pharmaceutically acceptable carrier; (b) administeringan effective dose of the composition to a subject, whereby aniontransport is altered in the subject.
 40. The method of claim 39, whereinthe subject is a mammal.
 41. The method of claim 40, wherein the mammalis a human.
 42. A method for identifying an anion exchanger modulator,the method comprising: (a) exposing a SLC26A6 polypeptide to one or moretest substances, wherein the SLC26A6 polypeptide comprises a mouseSLC26A6 polypeptide or a human SLC26A6a polypeptide; (b) assayingbinding of a test substance to the isolated SLC26A6 polypeptide; and (c)selecting a candidate substance that demonstrates specific binding tothe SLC26A6 polypeptide.
 43. The method of claim 42, wherein the mouseSLC26A6 polypeptide comprises: (a) a polypeptide of SEQ ID NO:6 or 8; or(b) a polypeptide encoded by a nucleic acid molecule of SEQ ID NO:5 or7.
 44. The method of claim 42, wherein the mouse SLC26A6a polypeptidecomprises: (a) a polypeptide of SEQ ID NO:2; or (b) a polypeptideencoded by a nucleic acid molecule of SEQ ID NO:1.
 45. An aniontransporter modulator identified by the method of claim
 42. 46. A methodfor modulating anion transport activity in a subject, the methodcomprising: (a) preparing a composition comprising a modulatoridentified according to the method of claim 42, and a pharmaceuticallyacceptable carrier; (b) administering an effective dose of thecomposition to a subject, whereby SLC26 function is altered in thesubject.
 47. The method of claim 46, wherein the subject is a mammal.48. The method of claim 47, wherein the subject is a human.
 49. A methodfor modulating a SLC26A6 polypeptide in a subject, the method comprisingadministering an effective amount of a SLC26A6 modulator to the subject,wherein the SLC26A6 modulator comprises a pH modifier.
 50. The method ofclaim 49, wherein the subject is a mammal.
 51. The method of claim 50,wherein the mammal is a human.
 52. An isolated nucleic acid encoding amouse SLC26A1 polypeptide.
 53. The isolated nucleic acid of claim 52,further comprising a nucleic acid encoding a polypeptide of SEQ IDNO:10.
 54. The isolated nucleic acid of claim 52, further comprising anucleic acid of SEQ ID NO:9.
 55. An isolated mouse SLC26A1 polypeptide.56. The isolated mouse SLC26A1 polypeptide of claim 55, furthercomprising: (a) a polypeptide encoded by a nucleic acid of SEQ ID NO:9;or (b) a polypeptide comprising an amino acid sequence of SEQ ID NO: 10.57. The mouse SLC26A1 polypeptide of claim 55, further comprising afunctional mouse SLC26A1 polypeptide.
 58. A system for recombinantexpression of a mouse SLC26A1 polypeptide, the system comprising: (a) amouse SLC26A1 polypeptide; and (b) a host cell comprising the mouseSLC26A1 polypeptide.
 59. A method for identifying a modulator of aSLC26A1 polypeptide, the method comprising: (a) providing a recombinantexpression system whereby a functional mouse SLC26A1 polypeptide isexpressed in a host cell; (b) providing a test substance to the systemof (a); (c) assaying a level or quality of SLC26A1 function in thepresence of the test substance; (d) comparing the level or quality ofSLC26A1 function in the presence of the test substance with a controllevel or quality of SLC26A1 function; and (e) identifying a testsubstance as an anion transport modulator by determining a level orquality of SLC26A1 function in the presence of the test substance assignificantly changed when compared to a control level or quality ofSLC26A1 function.
 60. The method of claim 57, wherein the mouse SLC26A1comprises: (a) a polypeptide encoded by a nucleic acid of SEQ ID NO:9;or (b) a polypeptide comprising an amino acid sequence of SEQ ID NO:10.61. An anion transporter modulator identified by the method of claim 59.62. A method for identifying an anion exchanger modulator, the methodcomprising: (a) exposing an isolated mouse SLC26A1 polypeptide to one ormore test substances; (b) assaying binding of a test substance to theisolated mouse SLC26A1 polypeptide; and (c) selecting a candidatesubstance that demonstrates specific binding to the SLC26A1 polypeptide.63. The method of claim 62, wherein the mouse SLC26A1 comprises: (a) apolypeptide encoded by a nucleic acid of SEQ ID NO:9; or (b) apolypeptide comprising an amino acid sequence of SEQ ID NO:10.
 64. Ananion transporter modulator identified by the method of claim
 62. 65. Amethod for activating a SLC26A1 polypeptide in a subject, the methodcomprising administering an effective amount of a SLC26A1 modulator tothe subject, wherein the SLC26A1 modulator comprises an impermeantanion.
 66. The method of claim 65, wherein the impermeant anioncomprises Cl⁻ or formate.
 67. A method for modulating a SLC26A2polypeptide in a subject, the method comprising administering aneffective amount of a SLC26A2 modulator to the subject, wherein theSLC26A2 modulator comprises a pH modifier.
 68. An isolated nucleic acidencoding a porcine SLC26A6 polypeptide.
 69. The isolated nucleic acid ofclaim 68, further comprising a nucleic acid encoding a polypeptide ofSEQ ID NO:91.
 70. The isolated nucleic acid of claim 68, furthercomprising a nucleic acid of SEQ ID NO:90.
 71. An isolated porcineSLC26A6 polypeptide.
 72. The isolated porcine SLC26A6 polypeptide ofclaim 71, further comprising: (a) a polypeptide encoded by a nucleicacid of SEQ ID NO:90; or (b) a polypeptide comprising an amino acidsequence of SEQ ID NO:91.
 73. An isolated nucleic acid encoding aXenopus laevis SLC26A6 polypeptide.
 74. The isolated nucleic acid ofclaim 73, further comprising a nucleic acid encoding a polypeptide ofSEQ ID NO:89.
 75. The isolated nucleic acid of claim 73, furthercomprising a nucleic acid of SEQ ID NO:88.
 76. An isolated Xenopuslaevis SLC26A6 polypeptide.
 77. The isolated Xenopus laevis SLC26A6polypeptide of claim 76, further comprising: (a) a polypeptide encodedby a nucleic acid of SEQ ID NO:88; or (b) a polypeptide comprising anamino acid sequence of SEQ ID NO:89.
 78. An isolated nucleic acidencoding a Xenopus laevis SLC26A1 polypeptide.
 79. The isolated nucleicacid of claim 78, further comprising a nucleic acid encoding apolypeptide of SEQ ID NO:87.
 80. The isolated nucleic acid of claim 78,further comprising a nucleic acid of SEQ ID NO:86.
 81. An isolatedXenopus laevis SLC26A1 polypeptide.
 82. The isolated Xenopus laevisSLC26A1 polypeptide of claim 81, further comprising: (a) a polypeptideencoded by a nucleic acid of SEQ ID NO:86; or (b) a polypeptidecomprising an amino acid sequence of SEQ ID NO:87.
 83. An isolatednucleic acid encoding a Xenopus laevis SLC26A4 polypeptide.
 84. Theisolated nucleic acid of claim 83, further comprising a nucleic acidencoding a polypeptide of one of SEQ ID NOs:81, 83, and
 85. 85. Theisolated nucleic acid of claim 83, further comprising a nucleic acid ofone of SEQ ID NOs:80, 82, and
 84. 86. An isolated Xenopus laevis SLC26A4polypeptide.
 87. The isolated Xenopus laevis SLC26A4 polypeptide ofclaim 86, further comprising: (a) a polypeptide encoded by a nucleicacid of one of SEQ ID NOs:80, 82, and 84; or (b) a polypeptidecomprising an amino acid sequence of one of SEQ ID NOs:81, 83, and 85.